Novel Plant Acyltransferases Specific for Long-Chained, Multiply Unsaturated Fatty Acids

ABSTRACT

The invention relates to a process for the production of long-chain polyunsaturated fatty acids in an organism by introducing, into the organism, nucleic acids coding for polypeptides with acyltransferase activity. These nucleic acid sequences, if appropriate together with further nucleic acid sequences coding for polypeptides of the fatty acid or lipid metabolism biosynthesis, can advantageously be expressed in the organism. Furthermore, the invention relates to a method for the production of oils and/or triacylglycerides with an elevated content of long-chain polyunsaturated fatty acids. The invention furthermore relates to the nucleic acid sequences, and constructs, vectors and organisms comprising the nucleic acid sequences. A further part of the invention relates to oils, lipids and/or fatty acids produced by the process according to the invention and to their use.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/844,938, filed Jul. 28, 2010, which is a divisional of U.S.application Ser. No. 10/552,013, filed Sep. 30, 2005, which is thenational stage application (under 35 U.S.C. 371) of PCT/EP2004/003224filed Mar. 26, 2004, which claims benefit to German application10314759.4 filed Mar. 31, 2003 and German application 10348996.7 filedOct. 17, 2003. The entire contents of each of these applications arehereby incorporated by reference herein.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)13478_(—)00005. The size ofthe text file is 410 KB, and the text file was created on Dec. 6, 2012.

FIELD OF THE INVENTION

The present invention relates to a process for the production oflong-chain polyunsaturated fatty acids in an organism by introducing,into the organism, nucleic acids which code for polypeptides withacyltransferase activity. These nucleic acid sequences, if appropriatetogether with further nucleic acid sequences which code for polypeptidesof the fatty acid or lipid metabolism biosynthesis, can advantageouslybe expressed in the organism. Furthermore, the invention relates to amethod for the production of oils and/or triacylglycerides with anelevated content of long-chain polyunsaturated fatty acids.

The invention furthermore relates to the nucleic acid sequences, nucleicacid constructs, vectors and organisms comprising the nucleic acidsequences according to the invention, vectors comprising the nucleicacid sequences and/or the nucleic acid constructs and to transgenicorganisms comprising the abovementioned nucleic acid sequences, nucleicacid constructs and/or vectors.

A further part of the invention relates to oils, lipids and/or fattyacids produced by the process according to the invention and to theiruse.

DESCRIPTION OF RELATED ART

Fatty acids and triacylglycerides have a multiplicity of applications inthe food industry, in animal nutrition, in cosmetics and in thepharmacological sector. Depending on whether they are free saturated orunsaturated fatty acids or else triacylglycerides with an elevatedcontent of saturated or unsaturated fatty acids, they are suitable forvery different applications. Polyunsaturated ω-3-fatty acids andω-6-fatty acids are therefore an important constituent in animal andhuman food. Owing to the present-day composition of human food, anaddition of polyunsaturated ω-3-fatty acids, which are preferentiallyfound in fish oils, to the food is particularly important. Thus, forexample, polyunsaturated fatty acids such as docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) or eicosapentaenoic acid (=EPA,C20:5^(Δ5,8,11,14,17)) are added to baby formula to improve thenutritional value. The unsaturated fatty acid DHA is said to have apositive effect on the development of the brain.

Hereinbelow, polyunsaturated fatty acids are referred to as PUFA, PUFAs,LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA, long chain polyunsaturated fatty acids LCPUFA).

The various fatty acids and triglycerides are mainly obtained frommicroorganisms such as Mortierella and Schizochytrium or fromoil-producing plants such as soybean, oilseed rape, algae such asCrypthecodinium or Phaeodactylum and others, where they are obtained, asa rule, in the form of their triacylglycerides(=triglycerides=triglycerols). However, they can also be obtained fromanimals, such as, for example, fish. The free fatty acids areadvantageously prepared by hydrolysis. Higher polyunsaturated fattyacids such as DHA, EPA, arachidonic acid (=ARA, C20:4^(Δ5,8,11,14)),dihomo-γ-linolenic acid (C20:3^(Δ8,11,14)) or docosapentaenoic acid(DPA, C22:5^(Δ7,10,13,16,19)) can not be isolated from oil crop plantssuch as oilseed rape, soybean, sunflower or safflower. Conventionalnatural sources of these fatty acids are fish such as herring, salmon,sardine, redfish, eel, carp, trout, halibut, mackerel, zander or tuna,or algae.

Depending on the intended use, oils with saturated or unsaturated fattyacids are preferred. In human nutrition, for example, lipids withunsaturated fatty acids, specifically polyunsaturated fatty acids, arepreferred. The polyunsaturated ω-3-fatty acids are said to have apositive effect on the cholesterol level in the blood and thus on thepossibility of preventing heart disease. The risk of heart disease,stroke or hypertension can be reduced markedly by adding these ω-3-fattyacids to the food. Also, ω-3-fatty acids have a positive effect oninflammatory, specifically on chronically inflammatory, processes inassociation with immunological diseases such as rheumatoid arthritis.They are therefore added to foodstuffs, specifically to dieteticfoodstuffs, or are employed in medicaments. ω-6-Fatty acids such asarachidonic acid tend to have a negative effect on these disorders inconnection with these rheumatic diseases on account of our usual dietaryintake.

ω-3- and ω-6-fatty acids are precursors of tissue hormones, known aseicosanoids, such as the prostaglandins, which are derived fromdihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, andof the thromoxanes and leukotrienes, which are derived from arachidonicacid and eicosapentaenoic acid. Eicosanoids (known as the PG₂ series)which are formed from ω-6-fatty acids generally promote inflammatoryreactions, while eicosanoids (known as the PG₃ series) from ω-3-fattyacids have little or no proinflammatory effect.

Owing to the positive characteristics of the polyunsaturated fattyacids, there has been no lack of attempts in the past to make availablegenes which are involved in the synthesis of fatty acids ortriglycerides for the production of oils in various organisms with amodified content of unsaturated fatty acids. Thus, WO 91/13972 and itsUS equivalent describe a Δ-9-desaturase. WO 93/11245 claims aΔ-15-desaturase and WO 94/11516 a Δ-12-desaturase. Further desaturasesare described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582,WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem.,265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huanget al., Lipids 34, 1999: 649-659. However, the biochemicalcharacterization of the various desaturases has been insufficient todate since the enzymes, being membrane-bound proteins, present greatdifficulty in their isolation and characterization (McKeon et al.,Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol.Biochem., 26, 1988: 777-792). As a rule, membrane-bound desaturases arecharacterized by being introduced into a suitable organism which issubsequently analyzed for enzyme activity by analyzing the startingmaterials and the products. Δ-6-Desaturases are described in WO93/06712, U.S. Pat. No. 5,614,393, WO 96/21022, WO 00/21557 and WO99/27111 and the application for the production in transgenic organismsis described in WO 98/46763, WO 98/46764 and WO 98/46765. In thiscontext, the expression of various desaturases and the formation ofpolyunsaturated fatty acids are also described and claimed in WO99/64616 or WO 98/46776. As regards the expression efficacy ofdesaturases and its effect on the formation of polyunsaturated fattyacids, it must be noted that the expression of a single desaturase asdescribed to date has only resulted in low contents of unsaturated fattyacids/lipids such as, for example, γ-linolenic acid and stearidonicacid. Moreover, a mixture of ω-3- and ω-6-fatty acids was obtained, as arule.

Especially suitable microorganisms for the production of PUFAs aremicroalgae such as Phaeodactylum tricornutum, Porphoridium species,Thraustochytrium species, Schizochytrium species or Crypthecodiniumspecies, ciliates such as Stylonychia or Colpidium, fungi such asMortierella, Entomophthora or Mucor and/or mosses such asPhyscomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen(1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22:1060-1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology73: 269-278). Strain selection has resulted in the development of anumber of mutant strains of the microorganisms in question which producea series of desirable compounds including PUFAs. However, the mutationand selection of strains with an improved production of a particularmolecule such as the polyunsaturated fatty acids is a time-consuming anddifficult process. This is why recombinant methods as described aboveare preferred whenever possible. However, only limited amounts of thedesired polyunsaturated fatty acids such as DPA, EPA or ARA can beproduced with the aid of the abovementioned microorganisms, and,depending on the microorganism used, these are generally obtained asfatty acid mixtures of, for example, EPA, DPA and DHA.

The biosynthesis of LCPUFAs and the incorporation of LCPUFAs intomembranes or triacylglycerides proceeds via various metabolic pathways(A. Abbadi et al. (2001) European Journal of Lipid Science & Technology103:106-113). In bacteria such as Vibrio, and microalgae, such asSchizochytrium, malonyl-CoA is converted into LCPUFAs via anLCPUFA-producing polyketide synthase (J. G. Metz et al. (2001) Science293: 290-293; WO 00/42195; WO 98/27203; WO 98/55625). In microalgae,such as Phaeodactylum, and mosses, such as Physcomitrella, unsaturatedfatty acids such as linoleic acid or linolenic acid are converted, inthe form of their acyl-CoAs, in a plurality of desaturation andelongation steps to give LCPUFAs (T. K. Zank et al. (2000) BiochemicalSociety Transactions 28: 654-658). In mammals, the biosynthesis of DHAcomprises a chain shortening via beta-oxidation, in addition todesaturation and elongation steps.

In microorganisms and lower plants, LCPUFAs are present eitherexclusively in the form of membrane lipids, as is the case inPhyscomitrella and Phaeodactylum, or in membrane lipids andtriacylglycerides, as is the case in Schizochytrium and Mortierella.Incorporation of LCPUFAs into lipids and oils is catalyzed by variousacyltransferases and transacylases. These enzymes are already known tocarry out the incorporation of saturated and unsaturated fatty acids [A.R. Slabas (2001) J. Plant Physiology 158: 505-513; M. Frentzen (1998)Fett/Lipid 100: 161-166); S. Cases et al. (1998) Proc. Nat. Acad. Sci.USA 95: 13018-13023]. The acyltransferases are enzymes of the “Kennedypathway”, which are located on the cytoplasmic side of the membranesystem of the endoplasmic reticulum, referred to as “ER” hereinbelow. ERmembranes may be isolated experimentally as “microsomal fractions” fromvarious organisms [D. S. Knutzon et al. (1995) Plant Physiology 109:999-1006; S. Mishra & Y. Kamisaka (2001) Biochemistry 355: 315-322; U.S.Pat. No. 5,968,791]. These ER-bound acyltransferases in the microsomalfraction use acyl-CoA as the activated form of fatty acids.Glycerol-3-phosphate acyltransferase, referred to as GPAT hereinbelow,catalyzes the incorporation of acyl groups at the sn-1 position ofglycerol-3-phosphate. 1-Acylglycerol-3-phosphate acyltransferase (E.C.2.3.1.51), also known as lysophosphatidic acid acyltransferase andreferred to as LPAAT hereinbelow, catalyzes the incorporation of acylgroups at the sn-2 position of lysophosphatidic acid, abbreviated as LPAhereinbelow. After dephosphorylation of phosphatidic acid byphosphatidic acid phosphatase, diacylglycerol acyltransferase, referredto as DAGAT hereinbelow, catalyzes the incorporation of acyl groups atthe sn-3 position of diacylglycerols. Apart from these Kennedy pathwayenzymes, further enzymes capable of incorporating acyl groups frommembrane lipids into triacylglycerides are involved in the incorporationof fatty acids into triacylglycerides, namely phospholipiddiacylglycerol acyltransferase, referred to as PDAT hereinbelow, andlysophosphatidylcholine acyltransferase, referred to as LPCAT. Otherenzymes too, such as lecithin cholesterol acyltransferase (LCAT) can beinvolved in the transfer of acyl groups from membrane lipids intotriacylglycerides.

In WO 98/54302, Tjoelker et al. disclose a human lysophosphatidic acidacyltransferase and its potential use for the therapy of diseases, as adiagnostic, and a method for identifying modulators of the human LPAAT.In WO 98/54303, Leung et al. describe mammalian lysophosphatidic acidacyltransferases. Moreover, Leung et al. disclose a method for screeningpharmaceutical compounds for use, for example, in the treatment ofinflammations.

Moreover, a multiplicity of acyltransferases with a wide range ofenzymatic functions have been described in the literature and patents;thus, for example, WO 98/55632 and WO 93/10241 describe fatty acidalcohol acyltransferases which are involved in wax synthesis. WO98/55631 describes a DAGAT (diacylglycerol acyltransferase) fromMortierella ramanniana and a wax synthase from jojoba which also hasDAGAT activity. Slabas et al. (WO 94/13814) disclose a membrane-boundsn2-specific acyltransferase which has a different selectivity in theincorporation of monounsaturated erucic acid for the sn2 position andthus makes possible an increased erucic acid yield in oilseed rape. WO96/24674 describes a corresponding enzyme or gene from Limnanthesdouglasii. In WO 95/27791, Davies et al. describe LPAATs which arespecific for medium-length fatty acids and incorporate these into thesn2 position of triglycerides. Further novel plant acyltransferasesequences which have been found via homology comparisons with sequencesfrom public databases are described by Lassner et al. (WO 00/18889).Information on the specific function of these acyltransferase sequencesor biochemical data on the corresponding enzymes cannot be found in WO00/18889.

The enzymic activity of an LPCAT was first described in rats [Land(1960) Journal of Biological Chemistry 235: 2233-2237]. A plastidicLPCAT isoform [Akermoun et al. (2000) Biochemical Society Transactions28: 713-715] and an ER-bound isoform [Tumaney and Rajasekharan (1999)Biochimica et Biophysica Acta 1439: 47-56; Fraser and Stobart,Biochemical Society Transactions (2000) 28: 715-7718] exist in plants.LPCAT is involved in the biosynthesis and transacylation ofpolyunsaturated fatty acids in animals as well as in plants [Stymne andStobart (1984) Biochem. J. 223: 305-314; Stymne and Stobart (1987) in‘The Biochemistry of Plants: a Comprehensive Treatise’, Vol. 9 (Stumpf,P. K. ed.) pp. 175-214, Academic Press, New York]. An important functionof LPCAT or, more generally, of an acyl-CoA:lysophospholipidacyltransferase, referred to as LPLAT hereinbelow, in theATP-independent synthesis of acyl-CoA from phospholipids has beendescribed by Yamashita et al. (2001; Journal of Biological Chemistry276: 26745-26752).

Despite a lot of biochemical data, no genes coding for LPCAT have beenidentified previously. Genes of various other plant acyltransferaseshave been isolated and are described in WO 00/18889 (Novel PlantAcyltransferases).

Higher plants comprise polyunsaturated fatty acids such as linoleic acid(C18:2) and linolenic acid (C18:3). ARA, EPA and DHA are found not atall in the seed oil of higher plants, or only in traces (E. Ucciani:Nouveau Dictionnaire des Huiles Véegétales. Technique &Documentation-Lavoisier, 1995. ISBN: 2-7430-0009-0). It is advantageousto produce LCPUFAs in higher plants, preferably in oil seeds such asoilseed rape, linseed, sunflower and soybean, since large amounts ofhigh-quality LCPUFAs for the food industry, animal nutrition andpharmaceutical purposes may be obtained at low costs in this way. Tothis end, it is advantageous to introduce into and express in oil seedsgenes coding for enzymes of the biosynthesis of LCPUFAs by geneticengineering methods. Said genes code, for example, for Δ-6-desaturase,Δ-6-elongase, Δ-5-desaturase, Δ-5-elongase and Δ-6-desaturase. Thesegenes may advantageously be isolated from microorganisms and lowerplants which produce LCPUFAs and incorporate them in the membranes ortriacylglycerides. Thus, Δ-6-desaturase genes have already been isolatedfrom the moss Physcomitrella patens and Δ-6-elongase genes have alreadybeen isolated from P. patens and the nematode C. elegans.

Transgenic plants which express genes coding for enzymes of LCPUFAbiosynthesis are suitable for producing small amounts of these LCPUFAs;however, there is the risk that the latter are incorporated not intotriacylglycerides, but into membranes, since the endogenousacyltransferases and transacylases may not recognize LCPUFAs assubstrate and, accordingly, do not incorporate them intotriacylglycerides. This is undesired for the following reasons: (i) themain lipid fraction in oil seeds are triacylglycerides. This is why, foreconomical reasons, it is necessary to concentrate LCPUFAs intriacylglycerides. LCPUFAs which are incorporated into membranes canmodify the physical characteristics of the membranes and thus haveharmful effects on the integrity and transport characteristics of themembranes and on the stress tolerance of plants.

First transgenic plants which comprise and express genes coding forenzymes of LCPUFA biosynthesis and produce LCPUFAs have been describedfor the first time, for example, in DE 102 19 203 (process for theproduction of polyunsaturated fatty acids in plants). However, theseplants produce LCPUFAs in amounts which require further optimization forprocessing the oils present in said plants.

In order to enable food and feed to be enriched with thesepolyunsaturated fatty acids, there is therefore a great need for asimple, inexpensive process for producing said polyunsaturated fattyacids, especially in eukaryotic systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows vector map of pSUN3CeLPLAT.

FIG. 2 shows amino acid sequence alignment of C. elegans LPLATs(Ce-T06E8.1 and Ce-F59F4.4) with the M. musculus LPAAT (Mm-NP061350).

FIG. 3 shows fatty acid profiles of transgenic C13ABYS86 S. cerevisiaecells.

FIG. 4 shows elongation of exogenously applied 18:2^(Δ9, 12) and18:3^(Δ9, 12, 15), respectively, following their endogenousΔ-6-desaturation (data from FIGS. 2 and 3).

FIG. 5 shows fatty acid profiles of transgenic C13ABYS86 S. cerevisiaecells.

FIG. 6 shows acyl-CoA composition of transgenic INVSc1 yeasts which hadbeen transformed with the vectors pESCLeu PpD6Pse1/pYes2 (A) orpESCLeu-PpD6-Pse1/pYes2-T06E8.1 (B).

FIG. 7 shows fatty acid profiles of transgenic INVSc1 S. cerevisiaecells.

FIG. 8 shows fatty acid profiles of transgenic INVSc1 S. cerevisiaecells.

FIG. 9A shows vector map of pGPTV LeB4-700+T06E8.1.

FIG. 9B shows vector map of pGPTV USP/OCS-1,2,3 PSE1(Pp)+D6-Des(Pt)+2AT(T06E8-1).

FIGS. 10A and 10B show biosynthetic pathway of LCPUFAs.

FIG. 11 shows comparison of GPAT and LPAAT substrate specificities inlinseed, sunflower and Mortierella alpine.

FIG. 12 shows comparison of LPCAT substrate specificity in linseed,sunflower and Mortierella alpine.

FIG. 13 shows alignment of SEQ ID NO: 2 with Swiss Prot database.

FIG. 14 shows alignment of SEQ ID NO: 5 with Swiss Prot database.

FIG. 15 shows alignment of SEQ ID NO: 35 with Swiss Prot database.

FIG. 16 shows alignment of SEQ ID NO: 23 with Swiss Prot database.

FIG. 17 shows alignment of SEQ ID NO: 27 with Swiss Prot database.

FIG. 18 shows alignment of SEQ ID NO: 8 with Swiss Prot database.

FIG. 19 shows alignment of SEQ ID NO: 10 with Swiss Prot database.

FIG. 20 shows alignment of SEQ ID NO: 12 with Swiss Prot database.

FIG. 21 shows Western blot analyses of the Thraustochytrium LPAATexpressed in E. coli as fusion protein (LPAAT-FP) with N-terminal GSTtag and C-terminal His tag (A) and acyl-CoA specificity of theThraustochytrium LPAAT expressed as GST fusion protein in E. coli (B).

FIG. 22 shows Western blot analysis of the Shewanella LPAAT expressed inE. coli as fusion protein with C-terminal His tag (A) and functionalexpression of the Shewanella LPAAT in E. coli (B).

FIG. 23 shows expression of Mortierella LPAAT (MaB4_AT) in yeast, andfeeding of 18:2 Δ9,12 fatty acids (A+B).

FIG. 24 shows expression of Mortierella LPAAT (MaB4_AT) in yeast, andfeeding of 18:3 Δ9,12,15 fatty acids (C+D).

FIG. 25 shows expression of Mortierella LPAAT (MaB4_AT) in yeast, andfeeding of 18:2 Δ9,12 fatty acids (A+B). Analysis of the neutral lipids.

FIG. 26 shows expression of Mortierella LPAAT (MaB4_AT) in yeast, andfeeding of 18:3 Δ9,12,15 fatty acids (C+D). Analysis of the neutrallipids.

DETAILED DESCRIPTION OF THE INVENTION

It was therefore the object to develop a process for the production ofpolyunsaturated fatty acids in an organism, advantageously in aeukaryotic organism, preferably in a plant. This object was achieved bythe process according to the invention for the production ofpolyunsaturated fatty acids in an organism, which comprises thefollowing steps:

-   a) introducing, into the organism, at least one nucleic acid    sequence with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ    ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,    SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID    NO: 20, which codes for a polypeptide with lysophosphatidic acid    acyltransferase activity; or-   b) introducing, into the organism, at least one nucleic acid    sequence with the sequence shown in SEQ ID NO: 22, SEQ ID NO: 24 or    SEQ ID NO: 26, which codes for a polypeptide with    glycerol-3-phosphate acyltransferase activity; or-   c) introducing, into the organism, at least one nucleic acid    sequence with the sequence shown in SEQ ID NO: 28, SEQ ID NO: 30 or    SEQ ID NO: 32 which codes for a polypeptide with diacylglycerol    acyltransferase activity; or-   d) introducing, into the organism, at least one nucleic acid    sequence with the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 36,    which codes for a polypeptide with lecithin cholesterol    acyltransferase activity; or-   e) introducing, into the organism, at least one nucleic acid    sequence which can be derived from the coding sequence in SEQ ID NO:    1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID    NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16,    SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID    NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34    or SEQ ID NO: 36 as the result of the degeneracy of the genetic    code, or-   f) introducing, into the organism, at least one derivative of the    nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID    NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ    ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:    20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ    ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36, which code    for polypeptides with the amino acid sequence shown in SEQ ID NO: 2,    SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:    15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ    ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:    33, SEQ ID NO: 35 or SEQ ID NO: 37 and which have at least 40%    homology at the amino acid level with SEQ ID NO: 2, SEQ ID NO: 5,    SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID    NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,    SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID    NO: 35 or SEQ ID NO: 37 and have an equivalent lysophosphatidic acid    acyltransferase activity, glycerol-3-phosphate acyltransferase    activity, diacylglycerol acyltransferase activity or lecithin    cholesterol acyltransferase activity, and-   g) culturing and harvesting the organism.

Advantageously, the polyunsaturated fatty acids produced in the processof the invention comprise at least two, advantageously three, four orfive, double bonds. The fatty acids particularly advantageously comprisefour or five double bonds. Fatty acids produced in the processadvantageously have 18, 20, 22 or 24 carbon atoms in the fatty acidchain; preferably, the fatty acids comprise 20, 22 or 24 carbon atoms inthe fatty acid chain. Advantageously, saturated fatty acids are reactedto a minor extent, or not at all, with the nucleic acids used in theprocess. A minor extent is understood as meaning that the saturatedfatty acids are reacted with less than 5%, advantageously less than 3%,especially advantageously with less than 2% of the activity incomparison with polyunsaturated fatty acids. These fatty acids which areproduced may be produced in the process as a single product or bepresent in a fatty acid mixture.

The nucleic acid sequences used in the process of the invention areisolated nucleic acid sequences which code for polypeptides withlysophosphatidic acid acyltransferase activity, glycerol-3-phosphateacyltransferase activity, diacylglycerol acyltransferase activity and/orlecithin cholesterol acyltransferase activity.

The polyunsaturated fatty acids produced in the process areadvantageously bound in membrane lipids and/or triacylglycerides but mayalso occur in the organisms as free fatty acids or else bound in theform of other fatty acid esters. In this context, they may be present asstated as “pure products” or else advantageously in the form of mixturesof various fatty acids or mixtures of different glycerides. The variousfatty acids bound in the triacylglycerides can be derived here fromshort-chain fatty acids having from 4 to 6 carbon atoms, medium-chainfatty acids having from 8 to 12 carbon atoms or long-chain fatty acidshaving from 14 to 24 carbon atoms, with preference being given to thelong-chain fatty acids and particular preference being given to thelong-chain fatty acids, LCPUFAs, of C₁₈-, C₂₀-, C₂₂- and/or C₂₄-fattyacids.

The process of the invention advantageously produces fatty acid esterswith polyunsaturated C₁₈-, C₂₀-, C₂₂- and/or C₂₄-fatty acid molecules,with at least two double bonds being present in the fatty acid ester.These fatty acid molecules preferably comprise three, four or fivedouble bonds and advantageously lead to the synthesis of hexadecadienoicacid (C16:2^(Δ9, 12)), γ-linolenic acid (=GLA, C18:3^(Δ6,9,12)),stearidonic acid (=SDA, C18:4^(Δ6,9,12,15)), dihomo-γ-linolenic acid(=DGLA, 20:3^(Δ8,11,14)), eicosatetraenoic acid (=ETA,C20:4^(Δ5,8,11,14)), arachidonic acid (ARA), eicosapentaenoic acid (EPA)or mixtures thereof, preferably EPA and/or ARA.

The fatty acid esters with polyunsaturated C₁₈-, C₂₀-, C₂₂- and/orC₂₄-fatty acid molecules can be isolated, from the organisms which havebeen used for the preparation of the fatty acid esters, in the form ofan oil or lipid, for example in the form of compounds such assphingolipids, phosphoglycerides, lipids, glycolipids such asglycosphingolipid, phospholipids such as phosphatidylethanolamine,phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides,diacylglycerides, triacylglycerides or other fatty acid esters such asthe acetyl-coenzyme A esters which comprise the polyunsaturated fattyacids with at least two, preferably three double bonds; advantageouslythey are isolated in the form of their diacylglycerides,triacylglycerides and/or in the form of phosphatidylcholine, especiallypreferably in the form of the triacylglycerides. In addition to theseesters, the polyunsaturated fatty acids are also present in theorganisms, advantageously the plants, as free fatty acids or bound inother compounds. As a rule, the various above-mentioned compounds (fattyacid esters and free fatty acids) are present in the organisms with anapproximate distribution of 80 to 90% by weight of triglycerides, 2 to5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 to5% by weight of free fatty acids, 2 to 8% by weight of phospholipids,the total of the various compounds amounting to 100% by weight.

The process according to the invention yields the LCPUFAs produced in acontent of at least 3% by weight, advantageously at least 5% by weight,preferably at least 8% by weight, especially preferably at least 10% byweight, most preferably at least 15% by weight, based on the total fattyacids in the transgenic organisms, advantageously in a transgenic plant.The fatty acids are advantageously produced in bound form. With the aidof the nucleic acids used in the process according to the invention,these unsaturated fatty acids can be brought into the sn1, sn2 and/orsn3 position of the triglycerides which are advantageously prepared.Since a plurality of reaction steps are performed by the startingcompounds hexadecadienoic acid (C16:2), linoleic acid (C18:2) andlinolenic acid (C18:3) in the process according to the invention, theend products of the process such as, for example, arachidonic acid (ARA)or eicosapentaenoic acid (EPA) are not obtained as absolutely pureproducts; minor traces of the precursors are always present in the endproduct. If, for example, both linoleic acid and linolenic acid arepresent in the starting organism and the starting plant, the endproducts such as ARA and EPA are present as mixtures. The precursorsshould advantageously not amount to more than 20% by weight, preferablynot to more than 15% by weight, especially preferably not to more than10% by weight, most preferably not to more than 5% by weight, based onthe amount of the end product in question. Advantageously, only ARA oronly EPA, bound or as free acids, are produced as end products in atransgenic plant in the process according to the invention.

If both compounds (ARA and EPA) are produced simultaneously, they areadvantageously produced in a ratio of at least 1:2 (EPA:ARA),advantageously of at least 1:3, preferably 1:4, especially preferably1:5.

Owing to the nucleic acid sequences according to the invention, anincrease in the yield of polyunsaturated fatty acids of at least 50%,advantageously of at least 80%, especially advantageously of at least100%, very especially advantageously of at least 150%, in comparisonwith the nontransgenic starting organism, can be obtained by comparisonin GC analysis (see examples). In a further advantageous embodiment, theyield of polyunsaturated fatty acids can be increased by at least 200%,preferably by at least 250%, very especially preferably by at least300%.

Chemically pure polyunsaturated fatty acids or fatty acid compositionscan also be synthesized by the processes described above. To this end,the fatty acids or the fatty acid compositions are isolated from theorganism, such as the microorganisms or the plants or the culture mediumin or on which the organisms have been grown, or from the organism andthe culture medium, in the known manner, for example via extraction,distillation, crystallization, chromatography or combinations of thesemethods. These chemically pure fatty acids or fatty acid compositionsare advantageous for applications in the food industry sector, thecosmetics industry sector and especially the pharmacological industrysector.

Suitable organisms for the production in the process according to theinvention are, in principle, any organisms such as microorganisms,nonhuman animals or plants. Advantageously the process according to theinvention employs transgenic organisms such as fungi, such asMortierella or Traustochytrium, yeasts such as Saccharomyces orSchizosaccharomyces, mosses such as Physcomitrella or Ceratodon,nonhuman animals such as Caenorhabditis, algae such as Crypthecodiniumor Phaeodactylum or plants such as dicotyledonous or monocotyledonousplants. Organisms which are especially advantageously used in theprocess according to the invention are organisms which belong to theoil-producing organisms, that is to say which are used for theproduction of oils, such as fungi, such as Mortierella orTraustochytrium, algae such as Crypthecodinium, Phaeodactylum, orplants, in particular plants, preferably oil crop plants which compriselarge amounts of lipid compounds, such as peanut, oilseed rape, canola,sunflower, safflower, poppy, mustard, hemp, castor-oil plant, olive,sesame, Calendula, Punica, evening primrose, verbascum, thistle, wildroses, hazelnut, almond, macadamia, avocado, bay, pumpkin/squash,linseed, soybean, pistachios, borage, trees (oil palm, coconut orwalnut) or arable crops such as maize, wheat, rye, oats, triticale,rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants suchas potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa orbushy plants (coffee, cacao, tea), Salix species, and perennial grassesand fodder crops. Preferred plants according to the invention are oilcrop plants such as peanut, oilseed rape, canola, sunflower, safflower,poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica,evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oilpalm, coconut). Especially preferred are plants which are high in C18:2-and/or C18:3-fatty acids, such as sunflower, safflower, tobacco,verbascum, sesame, cotton, pumpkin/squash, poppy, evening primrose,walnut, linseed, hemp, thistle or safflower. Very especially preferredplants are plants such as safflower, sunflower, poppy, evening primrose,walnut, linseed or hemp.

It is advantageous to the inventive process described to introduce, inaddition to the nucleic acids introduced in step (a) to (f) of theprocess, further nucleic acids which code for enzymes of the fatty acidor lipid metabolism into the organism.

In principle, all genes of the fatty acid or lipid metabolism can beused in the process for the production of polyunsaturated fatty acids,advantageously in combination with the inventiveacyl-CoA:lysophospholipid acyltransferase. Genes of the fatty acid orlipid metabolism selected from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:lysophospholipid acyltransferases, fatty acid synthase(s), fatty acidhydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme Aoxidase(s), fatty acid desaturase(s), fatty acid acetylenases,lipoxygenases, triacylglycerol lipases, alleneoxide synthases,hydroperoxide lyases or fatty acid elongase(s) are advantageously usedin combination with the acyl-CoA:lysophospholipid acyltransferase. Genesselected from the group of the acyl-CoA:lysophospholipidacyltransferases, Δ-4-desaturases, Δ-5-desaturases, Δ-6-desaturases,Δ-8-desaturases, Δ-9-desaturases, Δ-12-desaturases, Δ-5-elongases,Δ-6-elongases or Δ-9-elongases are especially preferably used incombination with the abovementioned genes for lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase, it beingpossible to use individual genes or a plurality of genes in combination.

Owing to the enzymatic activity of the nucleic acids used in the processaccording to the invention which code for polypeptides withlysophosphatidic acid acyltransferase glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase activity, advantageously in combination with nucleicacid sequences which code for polypeptides of the fatty acid or lipidmetabolism, such as the acyl-CoA:lysophospholipid acyltransferaseactivity, the Δ-4-, Δ-5-, Δ-6-, Δ-8-desaturase or the Δ-5-, Δ-6- orΔ-9-elongase activity, a wide range of polyunsaturated fatty acids canbe produced in the process according to the invention. Depending on thechoice of the organisms, such as the advantageous plant, used for theprocess according to the invention, mixtures of the variouspolyunsaturated fatty acids or individual polyunsaturated fatty acids,such as EPA or ARA, can be produced in free or bound form. Depending onthe prevailing fatty acid composition in the starting plant (C18:2- orC18:3-fatty acids), fatty acids which are derived from C18:2-fattyacids, such as GLA, DGLA or ARA, or fatty acids which are derived fromC18:3-fatty acids, such as SDA, ETA or EPA, are thus obtained. If onlylinoleic acid (=LA, C18:2^(Δ9, 12)) is present as unsaturated fatty acidin the plant used for the process, the process can only afford GLA, DGLAand ARA as products, all of which can be present as free fatty acids orin bound form. If only α-linolenic acid (=ALA, 8:3^(Δ9,12,15)) ispresent as unsaturated fatty acid in the plant used for the process, asis the case, for example, in linseed, the process can only afford SDA,ETA and EPA as products, all of which can be present as free fatty acidsor in bound form, as described above. By modifying the activity of theenzymes involved in the synthesis, lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase advantageouslyin combination with acyl-CoA: lysophospholipid acyltransferase, Δ-5-,Δ-6-desaturase and/or Δ-6-elongase or with acyl-CoA:lysophospholipidacyltransferase, Δ-5-, Δ-8-desaturase and/or Δ-9-elongase or incombination with only the first three genes, acyl-CoA: lysophospholipidacyltransferase, Δ-6-desaturase and/or Δ-6-elongase or acyl-CoA:lysophospholipid acyltransferase, Δ-8-desaturase and Δ-9-elongase, ofthe synthesis cascade, it is possible to produce, in a targeted fashion,only individual products in the abovementioned organisms, advantageouslyin the abovementioned plants. Owing to the activity of Δ-6-desaturaseand Δ-6-elongase, for example, GLA and DGLA, or SDA and ETA, are formed,depending on the starting plant and unsaturated fatty acid. DGLA or ETAor mixtures of these are preferably formed. If Δ-5-desaturase isadditionally introduced into the organisms, advantageously into theplant, ARA or EPA is additionally formed. This also applies to organismsinto which Δ-8-desaturase and Δ-9-elongase have been introducedpreviously. Advantageously, only ARA or EPA or mixtures of these aresynthesized, depending on the fatty acid present in the organism, or inthe plant, which acts as starting substance for the synthesis. Sincebiosynthetic cascades are involved, the end products in question are notpresent in pure form in the organisms. Small amounts of the precursorcompounds are always additionally present in the end product. Thesesmall amounts amount to less than 20% by weight, advantageously lessthan 15% by weight, especially advantageously less than 10% by weight,most advantageously less than 5, 4, 3, 2 or 1% by weight, based on theend product DGLA, ETA or their mixtures, or ARA, EPA or their mixtures.

To increase the yield in the described method for the production of oilsand/or triglycerides with an advantageously elevated content ofpolyunsaturated fatty acids, it is advantageous to increase the amountof starting product for the synthesis of fatty acids; this can beachieved for example by introducing, into the organism, a nucleic acidwhich codes for a polypeptide with Δ-12-desaturase. This is particularlyadvantageous in oil-producing organisms such as oilseed rape which arehigh in oleic acid. Since these organisms are only low in linoleic acid(Mikoklajczak et al., Journal of the American Oil Chemical Society, 38,1961, 678-681), the use of the abovementioned Δ-12-desaturases forproducing the starting material linoleic acid is advantageous.

Nucleic acids used in the process according to the invention areadvantageously derived from plants such as algae such as Isochrysis orCrypthecodinium, algae/diatoms such as Phaeodactylum, mosses such asPhyscomitrella or Ceratodon, or higher plants such as the Primulaceaesuch as Aleuritia, Calendula stellata, Osteospermum spinescens orOsteospermum hyoseroides, microorganisms such as fungi, such asAspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor orMortierella, bacteria such as Shewanella, yeasts or animals such asnematodes such as Caenorhabditis, insects or humans. The nucleic acidsare advantageously derived from fungi, animals, or from plants such asalgae or mosses, preferably from nematodes such as Caenorhabditis.

The process according to the invention advantageously employs theabovementioned nucleic acid sequences or their derivative or homologswhich code for polypeptides which retain the enzymatic activity of theproteins encoded by nucleic acid sequences. These sequences,individually or in combination with the nucleic acid sequences whichcode for lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase and/or lecithincholesterol acyltransferase are cloned into expression constructs andused for the introduction into, and expression in, organisms. Owing totheir construction, these expression constructs make possible anadvantageous optimal synthesis of the polyunsaturated fatty acidsproduced in the process according to the invention.

In a preferred embodiment, the process furthermore comprises the step ofobtaining a cell or an intact organism which comprises the nucleic acidsequences used in the process, where the cell and/or the organism istransformed with a nucleic acid sequence according to the inventionwhich codes for the lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferaseand/or lecithin cholesterol acyltransferase, a gene construct or avector as described below, alone or in combination with further nucleicacid sequences which code for proteins of the fatty acid or lipidmetabolism. In a further preferred embodiment, this process furthermorecomprises the step of obtaining the fine chemical from the culture. Theculture can, for example, take the form of a fermentation culture, forexample in the case of the cultivation of microorganisms, such as, forexample, Mortierella, Saccharomyces or Traustochytrium, or a greenhouse-or field-grown culture of a plant. The cell or the organism producedthus is advantageously a cell of an oil-producing organism, such as anoil crop plant, such as, for example, peanut, oilseed rape, canola,linseed, hemp, soybean, safflower, sunflowers or borage.

In the case of plant cells, plant tissue or plant organs, “growing” isunderstood as meaning, for example, the cultivation on or in a nutrientmedium, or of the intact plant on or in a substrate, for example in ahydroponic culture, potting compost or on arable land.

For the purposes of the invention, “transgenic” or “recombinant” means,with regard to the example of a nucleic acid sequence, an expressioncassette (=gene construct) or a vector comprising the nucleic acidsequence according to the invention or an organism transformed with thenucleic acid sequences, expression cassette or vector according to theinvention, all those constructions brought about by recombinant methodsin which either

-   a) the nucleic acid sequence according to the invention, or-   b) a genetic control sequence which is operably linked with the    nucleic acid sequence according to the invention, for example a    promoter, or-   c) (a) and (b)    are not located in their natural genetic environment or have been    modified by recombinant methods, it being possible for the    modification to take the form of, for example, a substitution,    addition, deletion, inversion or insertion of one or more nucleotide    residues. The natural genetic environment is understood as meaning    the natural genomic or chromosomal locus in the original organism or    the presence in a genomic library. In the case of a genomic library,    the natural genetic environment of the nucleic acid sequence is    preferably retained, at least in part. The environment flanks the    nucleic acid sequence at least on one side and has a sequence length    of at least 50 bp, preferably at least 500 bp, especially preferably    at least 1000 bp, most preferably at least 5000 bp. A naturally    occurring expression cassette—for example the naturally occurring    combination of the natural promoter of the inventive nucleic acid    sequences with the corresponding lysophosphatidic acid    acyltransferase, glycerol-3-phosphate acyltransferase,    diacylglycerol acyltransferase and/or lecithin cholesterol    acyltransferase genes—becomes a transgenic expression cassette when    this expression cassette is modified by nonnatural, synthetic    (“artificial”) methods such as, for example, mutagenic treatment.    Suitable methods are described, for example, in U.S. Pat. No.    5,565,350 or WO 00/15815.

A transgenic organism or transgenic plant for the purposes of theinvention is understood as meaning, as above, that the nucleic acidsused in the process are not at their natural locus in the genome of anorganism, it being possible for the nucleic acids to be expressedhomologously or heterologously. However, as mentioned, transgenic alsomeans that, while the nucleic acids according to the invention are attheir natural position in the genome of an organism, the sequence hasbeen modified with regard to the natural sequence, and/or that theregulatory sequences of the natural sequences have been modified.Transgenic is preferably understood as meaning the expression of thenucleic acids according to the invention at an unnatural locus in thegenome, i.e. homologous or, preferably, heterologous expression of thenucleic acids takes place. Preferred transgenic organisms are fungi suchas Mortierella, mosses such as Physcomitrella, algae such asCryptocodinium or plants such as the oil crop plants.

Suitable organisms or host organisms for the nucleic acids, theexpression cassette or the vector used in the process according to theinvention are, in principle, advantageously all organisms which arecapable of synthesizing fatty acids, specifically unsaturated fattyacids, and/or which are suitable for the expression of recombinantgenes. Examples which may be mentioned are plants such as Arabidopsis,Asteraceae such as Calendula or crop plants such as soybean, peanut,castor-oil plant, sunflower, maize, cotton, flax, oilseed rape, coconut,oil palm, safflower (Carthamus tinctorius) or cacao bean,microorganisms, such as fungi, for example the genus Mortierella,Thraustochytrium, Saprolegnia, or Pythium, bacteria, such as the genusEscherichia, or Shewanella, yeasts, such as the genus Saccharomyces,cyanobacteria, ciliates, algae or protozoans such as dinoflagellates,such as Crypthecodinium. Preferred organisms are those which arenaturally capable of synthesizing substantial amounts of oil, such asfungi, such as Mortierella alpina, Pythium insidiosum, or plants such assoybean, oilseed rape, coconut, oil palm, safflower, flax, hemp,castor-oil plant, Calendula, peanut, cacao bean or sunflower, or yeastssuch as Saccharomyces cerevisiae, with soybean, flax, oilseed rape,safflower, sunflower, Calendula, Mortierella or Saccharomyces cerevisiaebeing especially preferred. In principle, suitable host organisms are,in addition to the above-mentioned transgenic organisms, also transgenicanimals, advantageously nonhuman animals, for example C. elegans.

Further utilizable host cells are detailed in: Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990).

Expression strains which can be used, for example those with a lowerprotease activity, are described in: Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128.

These include plant cells and certain tissues, organs and parts ofplants in all their phenotypic forms such as anthers, fibers, roothairs, stalks, embryos, calli, cotyledons, petioles, harvested material,plant tissue, reproductive tissue and cell cultures which are derivedfrom the actual transgenic plant and/or can be used for giving rise tothe transgenic plant.

Transgenic plants which comprise the polyunsaturated fatty acidssynthesized in the process according to the invention can advantageouslybe marketed directly without there being any need for the oils, lipidsor fatty acids synthesized to be isolated. Plants for the processaccording to the invention are listed as meaning intact plants and allplant parts, plant organs or plant parts such as leaf, stem, seeds,root, tubers, anthers, fibers, root hairs, stalks, embryos, calli,cotyledons, petioles, harvested material, plant tissue, reproductivetissue and cell cultures which are derived from the transgenic plantand/or can be used for giving rise to the transgenic plant. In thiscontext, the seed comprises all parts of the seed such as the seedcoats, epidermal cells, seed cells, endosperm or embryonic tissue.However, the compounds produced in the process according to theinvention can also be isolated from the organisms, advantageouslyplants, in the form of their oils, fat, lipids and/or free fatty acids.Polyunsaturated fatty acids produced by this process can be obtained byharvesting the organisms, either from the crop in which they grow, orfrom the field. This can be done via pressing or extraction of the plantparts, preferably the plant seeds. In this context, the oils, fats,lipids and/or free fatty acids can be obtained by what is known ascold-beating or cold-pressing without applying heat by pressing. Toallow for greater ease of disruption of the plant parts, specificallythe seeds, they are previously comminuted, steamed or roasted. The seedswhich have been pretreated in this manner can subsequently be pressed orextracted with solvents such as warm hexane. The solvent is subsequentlyremoved again. In the case of microorganisms, the latter are, afterharvesting, for example extracted directly without further processingsteps or else, after disruption, extracted via various methods withwhich the skilled worker is familiar. In this manner, more than 96% ofthe compounds produced in the process can be isolated. Thereafter, theresulting products are processed further, i.e. refined. In this process,substances such as the plant mucilages and suspended matter are firstremoved. What is known as desliming can be effected enzymatically or,for example, chemico-physically by addition of acid such as phosphoricacid. Thereafter, the free fatty acids are removed by treatment with abase, for example sodium hydroxide solution. The resulting product iswashed thoroughly with water to remove the alkali remaining in theproduct and then dried. To remove the pigments remaining in the product,the products are subjected to bleaching, for example using fuller'searth or active charcoal. At the end, the product is deodorized, forexample using steam.

The PUFAs or LCPUFAs produced by this process are preferably C₁₈-, C₂₀-,C₂₂- or C₂₄-fatty acid molecules with at least two double bonds in thefatty acid molecule, preferably three, four, five or six double bonds.These C₁₈-, C₂₀-, C₂₂- or C₂₄-fatty acid molecules can be isolated fromthe organism in the form of an oil, a lipid or a free fatty acid.Suitable organisms are, for example, those mentioned above. Preferredorganisms are transgenic plants.

One embodiment of the invention is therefore oils, lipids or fatty acidsor fractions thereof which have been produced by the above-describedprocess, especially preferably oil, lipid or a fatty acid compositioncomprising PUFAs and being derived from transgenic plants.

A further embodiment according to the invention is the use of the oil,lipid, the fatty acids and/or the fatty acid composition in feedstuffs,foodstuffs, cosmetics or pharmaceuticals.

The term “oil”, “lipid” or “fat” is understood as meaning a fatty acidmixture comprising unsaturated or saturated, preferably esterified,fatty acid(s). The oil, lipid or fat is preferably high inpolyunsaturated free or, advantageously, esterified fatty acid(s), inparticular linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid,arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoicacid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoicacid. The content of unsaturated esterified fatty acids preferablyamounts to approximately 30%, a content of 50% is more preferred, acontent of 60%, 70%, 80% or more is even more preferred. For theanalysis, the fatty acid content can, for example, be determined by gaschromatography after converting the fatty acids into the methyl estersby transesterification. The oil, lipid or fat can comprise various othersaturated or unsaturated fatty acids, for example calendulic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid and the like.The content of the various fatty acids in the oil or fat can vary inparticular, depending on the starting organism.

The polyunsaturated fatty acids with advantageously at least two doublebonds which are produced in the process are, as described above, forexample sphingolipids, phosphoglycerides, lipids, glycolipids,phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol orother fatty acid esters.

Starting from the polyunsaturated fatty acids with advantageously atleast two double bonds, which acids have been prepared in the processaccording to the invention, the polyunsaturated fatty acids which arepresent can be liberated for example via treatment with alkali, forexample aqueous KOH or NaOH, or acid hydrolysis, advantageously in thepresence of an alcohol such as methanol or ethanol, or via enzymaticcleavage, and isolated via, for example, phase separation and subsequentacidification via, for example, H₂SO₄. The fatty acids can also beliberated directly without the above-described processing step.

After their introduction into an organism, advantageously a plant cellor plant, the nucleic acids used in the process can either be present ona separate plasmid or integrated into the genome of the host cell. Inthe case of integration into the genome, integration can be random orelse be effected by recombination such that the native gene is replacedby the copy introduced, whereby the production of the desired compoundby the cell is modulated, or by the use of a gene in trans, so that thegene is linked functionally with a functional expression unit whichcomprises at least one sequence which ensures the expression of a geneand at least one sequence which ensures the polyadenylation of afunctionally transcribed gene. The nucleic acids are advantageouslyintroduced into the organisms via multiexpression cassettes orconstructs for multiparallel expression, advantageously into the plantsfor the multiparallel seed-specific expression of genes.

Mosses and algae are the only known plant systems which producesubstantial amounts of polyunsaturated fatty acids such as arachidonicacid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoicacid (DHA). Mosses comprise PUFAs in membrane lipids, while algae,organisms which are related to algae and a few fungi also accumulatesubstantial amounts of PUFAs in the triacylglycerol fraction. This iswhy nucleic acid molecules are suitable which are isolated from suchstrains which also accumulate PUFAs in the triacylglycerol fraction,particularly advantageously for the process according to the inventionand thus for the modification of the lipid and PUFA production system ina host, in particular plants such as oil crop plants, for exampleoilseed rape, canola, linseed, hemp, soybeans, sunflowers and borage.They can therefore be used advantageously in the process according tothe invention.

Substrates of the nucleic acids used in the process according to theinvention which code for polypeptides with lysophosphatidic acidacyltransferase activity, glycerol-3-phosphate acyltransferase activity,diacylglycerol acyltransferase activity or lecithin cholesterolacyltransferase activity, and/or of the further nucleic acids used, suchas the nucleic acids which code for polypeptides of the fatty acidmetabolism or lipid metabolism selected from the group consisting ofacyl-CoA dehydrogenase(s), acyl-ACP[=acyl carrier protein]desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl coenzyme A carboxylase(s), acylcoenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allene oxidesynthase(s), hydroperoxide lyase(s) or fatty acid elongase(s) which areadvantageously suitable are C₁₆-, C₁₈-, C₂₀- or C₂₂-fatty acids. Thefatty acids converted in the process in the form of substrates arepreferably converted in the form of their acyl-CoA esters.

To produce the long-chain PUFAs according to the invention, thepolyunsaturated C₁₆- or C₁₈-fatty acids must first be desaturated by theenzymatic activity of a desaturase and subsequently be elongated by atleast two carbon atoms via an elongase. After one elongation cycle, thisenzyme activity gives C₁₈- or C₂₀-fatty acids and after two or threeelongation cycles C₂₂- or C₂₄-fatty acids. The activity of thedesaturases and elongases used in the process according to the inventionpreferably leads to C₁₈-, C₂₀-, C₂₂- and/or C₂₄-fatty acids,advantageously with at least two double bonds in the fatty acidmolecule, preferably with three, four or five double bonds, especiallypreferably to give C₂₀- and/or C₂₂-fatty acids with at least two doublebonds in the fatty acid molecule, preferably with three, four or fivedouble bonds in the molecule. After a first desaturation and theelongation have taken place, further desaturation steps such as, forexample, one in the Δ5 position may take place. Products of the processaccording to the invention which are especially preferred aredihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid,docosapentaenoic acid and/or docosahexaenoic acid. The C₁₈-fatty acidswith at least two double bonds in the fatty acid can be elongated by theenzymatic activity according to the invention in the form of the freefatty acid or in the form of the esters, such as phospholipids,glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol,diacylglycerol or triacylglycerol.

The preferred biosynthesis site of fatty acids, oils, lipids or fats inthe plants which are advantageously used is, for example, in general theseed or cell strata of the seed, so that seed-specific expression of thenucleic acids used in the process makes sense. However, it is obviousthat the biosynthesis of fatty acids, oils or lipids need not be limitedto the seed tissue, but can also take place in a tissue-specific mannerin all the other parts of the plant, for example in epidermal cells orin the tubers.

If microorganisms such as yeasts, such as Saccharomyces orSchizosaccharomyces, fungi such as Mortierella, Aspergillus,Phytophtora, Entomophthora, Mucor or Thraustochytrium, algae such asIsochrysis, Phaeodactylum or Crypthecodinium are used as organisms inthe process according to the invention, these organisms areadvantageously grown in fermentation cultures.

Owing to the use of the nucleic acids according to the invention whichcode for a lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase and/or lecithincholesterol acyltransferase, the polyunsaturated fatty acids produced inthe process can be increased by at least 5%, preferably by at least 10%,especially preferably by at least 20%, very especially preferably by atleast 50% in comparison with the wild type of the organisms which do notcomprise the nucleic acids recombinantly.

In principle, the polyunsaturated fatty acids produced by the processaccording to the invention in the organisms used in the process can beincreased in two different ways. Advantageously, the pool of freepolyunsaturated fatty acids and/or the content of the esterifiedpolyunsaturated fatty acids produced via the process can be enlarged.Advantageously, the pool of esterified polyunsaturated fatty acids inthe transgenic organisms is enlarged by the process according to theinvention.

If microorganisms are used as organisms in the process according to theinvention, they are grown or cultured in the manner with which theskilled worker is familiar, depending on the host organism. As a rule,microorganisms are grown in a liquid medium comprising a carbon source,usually in the form of sugars, a nitrogen source, usually in the form oforganic nitrogen sources such as yeast extract or salts such as ammoniumsulfate, trace elements such as salts of iron, manganese and magnesiumand, if appropriate, vitamins, at temperatures of between 0° C. and 100°C., preferably between 10° C. and 60° C., while gassing in oxygen. ThepH of the liquid medium can either be kept constant, that is to sayregulated during the culturing period, or not. The cultures can be grownbatchwise, semibatchwise or continuously. Nutrients can be provided atthe beginning of the fermentation or fed in semicontinuously orcontinuously. The polyunsaturated fatty acids produced can be isolatedfrom the organisms as described above by processes known to the skilledworker, for example by extraction, distillation, crystallization, ifappropriate precipitation with salt, and/or chromatography. To this end,the organisms can advantageously be disrupted beforehand.

If the host organisms are microorganisms, the process according to theinvention is advantageously carried out at a temperature of between 0°C. and 95° C., preferably between 10° C. and 85° C., especiallypreferably between 15° C. and 75° C., very especially preferably between15° C. and 45° C.

In this process, the pH value is advantageously kept between pH 4 and12, preferably between pH 6 and 9, especially preferably between pH 7and 8.

The process according to the invention can be operated batchwise,semibatchwise or continuously. An overview of known cultivation methodscan be found in the textbook by Chmiel (Bioprozeβtechnik 1. Einfführungin die Bioverfahrenstechnik [Bioprocess technology 1. Introduction toBioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren and periphere Einrichtungen[Bioreactors and peripheral equipment] (Vieweg Verlag,Brunswick/Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of thestrains in question. Descriptions of culture media for variousmicroorganisms can be found in the textbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

As described above, these media which can be employed in accordance withthe invention usually comprise one or more carbon sources, nitrogensources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch or cellulose. Sugars can also beadded to the media via complex compounds such as molasses or otherby-products from sugar refining. The addition of mixtures of a varietyof carbon sources may also be advantageous. Other possible carbonsources are oils and fats such as, for example, soya oil, sunflower oil,peanut oil and/or coconut fat, fatty acids such as, for example,palmitic acid, stearic acid and/or linoleic acid, alcohols and/orpolyalcohols such as, for example, glycerol, methanol and/or ethanol,and/or organic acids such as, for example, acetic acid and/or lacticacid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcescomprise ammonia in liquid or gaseous form or ammonium salts such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate or ammonium nitrate, nitrates, urea, amino acids or complexnitrogen sources such as cornsteep liquor, soya meal, soya protein,yeast extract, meat extract and others. The nitrogen sources can be usedindividually or as a mixture.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphorus and sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogenphosphate or dipotassiumhydrogenphosphate or the corresponding sodium-containing salts may beused as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention for culturingmicroorganisms usually also comprise other growth factors such asvitamins or growth promoters, which include, for example, biotin,riboflavin, thiamine, folic acid, nicotinic acid, panthothenate andpyridoxine. Growth factors and salts are frequently derived from complexmedia components such as yeast extract, molasses, cornsteep liquor andthe like. It is moreover possible to add suitable precursors to theculture medium. The exact composition of the media compounds heavilydepends on the particular experiment and is decided upon individuallyfor each specific case. Information on the optimization of media can befound in the textbook “Applied Microbiol. Physiology, A PracticalApproach” (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp.53-73, ISBN 0 19 963577 3). Growth media can also be obtained fromcommercial suppliers, for example Standard 1 (Merck) or BHI (brain heartinfusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by filter sterilization. The components may besterilized either together or, if required, separately. All mediacomponents may be present at the start of the cultivation or addedcontinuously or batchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° C., and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, ambientair into the culture. The temperature of the culture is normally 20° C.to 45° C. and preferably 25° C. to 40° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyachieved within 10 to 160 hours.

The fermentation broths obtained in this way, in particular thosecomprising polyunsaturated fatty acids, usually contain a dry mass offrom 7.5 to 25% by weight.

The fermentation broth can then be processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth. It is advantageous to process thebiomass after its separation.

However, the fermentation broth can also be thickened or concentratedwithout separating the cells, using known methods such as, for example,with the aid of a rotary evaporator, thin-film evaporator, falling-filmevaporator, by reverse osmosis or by nanofiltration. Finally, thisconcentrated fermentation broth can be processed to obtain the fattyacids present therein.

The fatty acids obtained in the process are also suitable as startingmaterial for the chemical synthesis of further products of interest. Forexample, they can be used in combination with one another or alone forthe preparation of pharmaceuticals, foodstuffs, animal feeds orcosmetics.

The invention furthermore relates to isolated nucleic acid sequencescoding for polypeptides having lysophosphatidic acid acyltransferaseactivity, glycerol-3-phosphate acyltransferase activity, diacylglycerolacyltransferase activity or lecithin cholesterol acyltransferaseactivity, wherein the lysophosphatidic acid acyltransferases,glycerol-3-phosphate acyltransferases, diacylglycerol acyltransferasesand/or lecithin cholesterol acyltransferases encoded by the nucleic acidsequences specifically convert C₁₈-, C₂₀-, C₂₂- or C₂₄-fatty acids withat least one double bonds in the fatty acid molecule and advantageouslyultimately incorporate these into diacylglycerides and/ortriacylglycerides.

Advantageous isolated nucleic acid sequences are sequences selected fromthe group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:    9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ    ID NO: 18 or SEQ ID NO: 20,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ    ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:    16, SEQ ID NO: 18 or SEQ ID NO: 20,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:    9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ    ID NO: 18 or SEQ ID NO: 20 which code for polypeptides with the    amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:    8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ    ID NO: 19 or SEQ ID NO: 21 and which have at least 40% homology at    the amino acid level with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8,    SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID    NO: 19 or SEQ ID NO: 21 and have lysophosphatidic acid    acyltransferase activity.

Further advantageous isolated nucleic acid sequences according to theinvention are sequences selected from the group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 22,    SEQ ID NO: 24 or SEQ ID NO: 26,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 22, SEQ ID NO: 24 or SEQ ID NO: 26,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 22,    SEQ ID NO: 24 or SEQ ID NO: 26, which code for polypeptides with the    amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID    NO: 27 and have at least 40% homology at the amino acid level with    SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 and have    glycerol-3-phosphate acyltransferase activity.

Additional advantageous isolated nucleic acid sequences according to theinvention are sequences selected from the group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 28,    SEQ ID NO: 30 or SEQ ID NO: 32,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 28,    SEQ ID NO: 30 or SEQ ID NO: 32, which code for polypeptides with the    amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID    NO: 33 and have at least 40% homology at the amino acid level with    SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33 and which have    diacylglycerol acyltransferase activity.

A further group of advantageous isolated nucleic acid sequencesaccording to the invention are sequences selected from the groupconsisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 34    or SEQ ID NO: 36,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 34 or SEQ ID NO: 36,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 34    or SEQ ID NO: 36, which code for polypeptides with the amino acid    sequence shown in SEQ ID NO: 35 or SEQ ID NO: 37 and which have at    least 40% homology at the amino acid level with SEQ ID NO: 35 or SEQ    ID NO: 37 and have lecithin cholesterol acyltransferase activity.

With the aid of these isolated nucleic acids according to the invention,LCPUFAs can be incorporated, in LCPUFA-producing organisms, at allpositions of, for example, a triacylglycerol, as indicated by theposition analyses of the lipids from LCPUFA-producing organisms.

The abovementioned isolated nucleic acid sequences according to theinvention can advantageously be combined with the following nucleic acidsequences, which code for polypeptides with acyl-CoA:lysophospholipidacyltransferase activity, selected from the group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 39,    SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45,-   b) nucleic acid sequences which can be derived from the coding    sequence present in SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or    SEQ ID NO: 45 as the result of the degeneracy of the genetic code,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 39,    SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45, which code for    polypeptides with the amino acid sequence shown in SEQ ID NO: 40,    SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46 and which have at    least 40% homology at the amino acid level with SEQ ID NO: 40, SEQ    ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46 and which have an    acyl-CoA: lysophospholipid acyltransferase activity.

All of the nucleic acid sequences used in the process according to theinvention are advantageously derived from a eukaryotic organism.

The nucleic acid sequences used in the process which code for proteinswith lyso-phosphatidic acid acyltransferase activity,glycerol-3-phosphate acyltransferase activity, diacylglycerolacyltransferase activity or lecithin cholesterol acyltransferaseactivity or for proteins of the fatty acid or lipid metabolism,advantageously for proteins with acyl-CoA:lysophospholipidacyltransferase, Δ-4-desaturase, Δ-5-desaturase, Δ-6-desaturase,Δ-8-desaturase, Δ-9-desaturase, Δ-12-desaturase, Δ-5-elongase,Δ-6-elongase or Δ-9-elongase activity are, advantageously alone orpreferably in combination, introduced in an expression cassette(=nucleic acid construct) which makes possible the expression of thenucleic acids in an organism, advantageously a plant or a microorganism.

To introduce the nucleic acids used in the process, the latter areadvantageously amplified and ligated in the known manner. Preferably, aprocedure following the protocol for Pfu DNA polymerase or a Pfu/Taq DNApolymerase mixture is followed. The primers are selected taking intoconsideration the sequence to be amplified. The primers shouldexpediently be chosen in such a way that the amplificate comprises theentire codogenic sequence from the start codon to the stop codon. Afterthe amplification, the amplificate is expediently analyzed. For example,a gel-electrophoretic separation can be carried out with regards toquality and quantity. Thereafter, the amplificate can be purifiedfollowing a standard protocol (for example Qiagen). An aliquot of thepurified amplificate is then available for the subsequent cloning step.Suitable cloning vectors are generally known to the skilled worker.These include, in particular, vectors which are capable of replicationin microbial systems, that is to say mainly vectors which ensureefficient cloning in yeasts or fungi and which make possible the stabletransformation of plants. Those which must be mentioned in particularare various binary and cointegrated vector systems which are suitablefor the T-DNA-mediated transformation. Such vector systems are, as arule, characterized in that they comprise at least the vir genesrequired for the Agrobacterium-mediated transformation and theT-DNA-delimiting sequences (T-DNA border). These vector systemspreferably also comprise further cis-regulatory regions such aspromoters and terminators and/or selection markers, by means of whichsuitably transformed organisms can be identified. While in the case ofcointegrated vector systems vir genes and T-DNA sequences are arrangedon the same vector, binary systems are based on at least two vectors,one of which bears vir genes, but no T-DNA, while a second one bearsT-DNA, but no vir gene. Owing to this fact, the last-mentioned vectorsare relatively small, easy to manipulate and to replicate both in E.coli and in Agrobacterium. These binary vectors include vectors from theseries pBIB-HYG, pPZP, pBecks, pGreen. In accordance with the invention,Bin19, pBI101, pBinAR, pGPTV and pCAMBIA are used by preference. Anoverview of binary vectors and their use is found in Hellens et al.,Trends in Plant Science (2000) 5, 446-451. In order to prepare thevectors, the vectors can first be linearized with restrictionendonuclease(s) and then modified enzymatically in a suitable manner.Thereafter, the vector is purified, and an aliquot is employed for thecloning step. In the cloning step, the enzymatically cleaved and, ifappropriate, purified amplificate is cloned using vector fragments whichhave been prepared in a similar manner, using ligase. In this context, aparticular nucleic acid construct, or vector or plasmid construct, canhave one or else more than one codogenic gene segment. The codogenicgene segments in these constructs are preferably linked functionallywith regulatory sequences. The regulatory sequences include, inparticular, plant sequences such as the above-described promoters andterminators. The constructs can advantageously be stably propagated inmicroorganisms, in particular in Escherichia coli and Agrobacteriumtumefaciens, under selective conditions and make possible the transferof heterologous DNA into plants or microorganisms.

The nucleic acids used in the process, the inventive nucleic acids andnucleic acid constructs, can be introduced into organisms such asmicroorganisms or advantageously plants, advantageously using cloningvectors, and thus be used in the transformation of plants such as thosewhich are published and cited in: Plant Molecular Biology andBiotechnology (CRC Press, Boca Raton, Fla.), Chapter 6/7, pp. 71-119(1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu.Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225. Thus, thenucleic acids, the inventive nucleic acids and nucleic acid constructs,and/or vectors used in the process can be used for the recombinantmodification of a broad spectrum of organisms, advantageously plants, sothat the latter become better and/or more efficient PUFA producers.

A series of mechanisms exists by which the modification of alysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase, or lecithin cholesterolacyltransferase protein according to the invention can influencedirectly the yield, production and/or production efficiency of a finechemical from an oil crop plant or a microorganism, owing to a modifiedprotein. The number or activity of the lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase, or lecithin cholesterol acyltransferase protein or geneand also of gene combinations of acyl-CoA:lysophospholipidacyltransferases, desaturases and/or elongases for example may haveincreased, so that greater amounts of the compounds produced areproduced de novo, since the organisms lacked this activity and abilityto biosynthesize prior to introduction of the corresponding gene(s).This applies analogously to the combination with further desaturases orelongases or further enzymes of the fatty acid and lipid metabolism. Theuse of various divergent sequences, i.e. sequences which differ at theDNA sequence level, may also be advantageous in this context, or elsethe use of promoters for gene expression which makes possible adifferent gene expression in the course of time, for example as afunction of the degree of maturity of a seed or an oil-storing tissue.

Owing to the introduction of a lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase,lecithin cholesterol acyltransferase, acyl-CoA: lysophospholipidacyltransferase, desaturase and/or elongase gene or morelysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase, lecithin cholesterolacyltransferase, acyl-CoA:lysophospholipid acyltransferase, desaturaseand/or elongase genes into an organism, alone or in combination withother genes in a cell, it is not only possible to increase biosynthesisflux towards the end product, but also to increase, or to create denovo, the corresponding triacylglycerol composition. Likewise, thenumber or activity of other genes which are involved in the import ofnutrients which are required for the biosynthesis of one or more finechemicals (e.g. fatty acids, polar and neutral lipids), can beincreased, so that the concentration of these precursors, cofactors orintermediates within the cells or within the storage compartment isincreased, whereby the ability of the cells to produce PUFAs asdescribed below is enhanced further. Fatty acids and lipids arethemselves desirable as fine chemicals; by optimizing the activity orincreasing the number of one or more lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase, lecithin cholesterol acyltransferase, acyl-CoA:lysophospholipid acyltransferase, desaturase and/or elongase genes whichare involved in the biosynthesis of these compounds, or by destroyingthe activity of one or more genes which are involved in the degradationof these compounds, an enhanced yield, production and/or efficiency ofproduction of fatty acid and lipid molecules from organisms,advantageously from plants, is made possible.

The isolated nucleic acid molecules used in the process according to theinvention code for proteins or parts of these, where the proteins or theindividual protein or parts thereof comprise(s) an amino acid sequencewith sufficient homology to an amino acid sequence of the sequence SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35 or SEQ ID NO: 37, so that the protein or part thereofhave a and retains an equivalent lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase orlecithin cholesterol acyltransferase activity. The protein or partthereof which is encoded by the nucleic acid molecule preferably retainsits essential enzymatic activity and the ability to participate in themetabolism of compounds required for the synthesis of cell membranes orlipid bodies in organisms, advantageously in plants, or in the transportof molecules across these membranes. Advantageously, the protein encodedby the nucleic acid molecules is at least approximately 40%, preferablyat least approximately 60% and more preferably at least approximately70%, 80% or 90% and most preferably at least approximately 95%, 96%,97%, 98%, 99% or more homologous to an amino acid sequence of thesequence SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37. For the purposes of theinvention homology or homologous are to be understood as meaningidentity or identical.

Essential enzymatic activity of the inventive lysophosphatidic acidacyltransferases, glycerol-3-phosphate acyltransferases, diacylglycerolacyltransferases or lecithin cholesterol acyltransferases used isunderstood as meaning that they retain at least an enzymatic activity ofat least 10%, preferably 20%, especially preferably 30% and veryespecially 40% in comparison with the proteins/enzymes encoded by thesequence with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34 or SEQ ID NO: 36 and their derivatives and can thus participatein the metabolism of compounds required for the synthesis of fattyacids, fatty acid esters such as diacylglycerides and/ortriacylglycerides in an organism, advantageously a plant cell, or in thetransport of molecules across membranes, meaning desaturated C₁₈-, C₂₀-,C₂₂- or C₂₄-carbon chains in the fatty acid molecule with double bondsat at least two, advantageously three, four or five positions.

Nucleic acids which can advantageously be used in the process arederived from bacteria, fungi or plants such as algae or mosses, such asthe genera Shewanella, Physcomitrella, Thraustochytrium, Fusarium,Phytophtora, Ceratodon, Isochrysis, Aleurita, Muscarioides, Mortierella,Borago, Phaeodactylum, Crypthecodinium or from nematodes such asCaenorhabditis, specifically from the genera and species Shewanellahanedai, Physcomitrella patens, Phytophtora infestans, Fusariumgraminaeum, Cryptocodinium cohnii, Ceratodon purpureus, Isochrysisgalbana, Aleurita farinosa, Muscarioides viallii, Mortierella alpina,Borago officinalis, Phaeodactylum tricornutum, or especiallyadvantageously from Caenorhabditis elegans.

Alternatively, the isolated nucleotide sequences used may code forlysophosphatidic acid acyltransferases, glycerol-3-phosphateacyltransferases, diacylglycerol acyltransferases or lecithincholesterol acyltransferases which hybridize with a nucleotide sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 orSEQ ID NO: 36, for example under stringent conditions.

The nucleic acid sequences used in the process are advantageouslyintroduced into an expression cassette which makes possible theexpression of the nucleic acids in organisms such as microorganisms orplants.

In doing so, the nucleic acid sequences which code for thelysophosphatidic acid acyltransferases, glycerol-3-phosphateacyltransferases, diacylglycerol acyltransferases or lecithincholesterol acyltransferases of the invention, and the nucleic acidsequences which code for the acyl-CoA:lysophospholipid acyltransferasesused in combination, the desaturases and/or the elongases are linkedfunctionally with one or more regulatory signals, advantageously forenhancing gene expression. These regulatory sequences are intended tomake possible the specific expression of the genes and proteins.Depending on the host organism, this may mean, for example, that thegene is expressed and/or overexpressed only after induction has takenplace, or else that it expresses and/or overexpresses immediately. Forexample, these regulatory sequences take the form of sequences to whichinductors or repressors bind, thus controlling the expression of thenucleic acid. In addition to these novel regulatory sequences, orinstead of these sequences, the natural regulation of these sequencesmay still be present before the actual structural genes and, ifappropriate, may have been genetically modified in such a way thatnatural regulation has been eliminated and expression of the genes hasbeen enhanced. However, the expression cassette (=expressionconstruct=gene construct) can also be simpler in construction, that isto say no additional regulatory signals have been inserted before thenucleic acid sequence or its derivatives, and the natural promotertogether with its regulation has not been removed. Instead, the naturalregulatory sequence has been mutated in such a way that regulation nolonger takes place and/or gene expression is enhanced. These modifiedpromoters can also be positioned on their own before the natural gene inthe form of part-sequences (=promoter with parts of the nucleic acidsequences of the invention) in order to enhance the activity. Moreover,the gene construct may advantageously also comprise one or more of whatare known as enhancer sequences in functional linkage with the promoter,which make possible an enhanced expression of the nucleic acid sequence.Additional advantageous sequences, such as further regulatory elementsor terminators, may also be inserted at the 3′ end of the DNA sequences.The lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase genes and the advantageously usedacyl-CoA:lysophospholipid acyltransferase, Δ-4-desaturase,Δ5-desaturase, Δ-6-desaturase and/or Δ-8-desaturase genes and/orΔ-5-elongase, Δ-6-elongase and/or Δ-9-elongase genes may be present inone or more copies in the expression cassette (=gene construct).Preferably, only one copy of the genes is present in each expressioncassette. This gene construct or the gene constructs can be expressedtogether in the host organism. In this context, the gene construct(s)can be inserted in one or more vectors and be present in the cell infree form, or else be inserted in the genome. It is advantageous for theinsertion of further genes in the host genome when the genes to beexpressed are present together in one gene construct.

In this context, the regulatory sequences or factors can, as describedabove, preferably have a positive effect on the gene expression of thegenes introduced, thus enhancing it. Thus, an enhancement of theregulatory elements, advantageously at the transcriptional level, maytake place by using strong transcription signals such as promotersand/or enhancers. In addition, however, enhanced translation is alsopossible, for example by improving the stability of the mRNA.

A further embodiment of the invention is one or more gene constructswhich comprise one or more sequences which are defined by SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 orits derivatives and which code for polypeptides as shown in SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35 or SEQ ID NO: 37. The abovementioned lysophosphatidic acidacyltransferases, glycerol-3-phosphate acyltransferases, diacylglycerolacyltransferases or lecithin cholesterol acyltransferases leadadvantageously to an exchange or incorporation of fatty acids betweenthe mono-, di- and/or triglyceride pool of the cell and the CoA-fattyacid ester pool, the substrate advantageously having one, two, three,four or five double bonds and advantageously 18, 20, 22 or 24 carbonatoms in the fatty acid molecule. The same applies to their homologs,derivatives or analogs, which are linked functionally with one or moreregulatory signals, advantageously for enhancing gene expression.

Advantageous regulatory sequences for the novel process are present forexample in promoters such as the cos, tac, trp, tet, trp-tet, Ipp, lac,Ipp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL promoterand are advantageously employed in Gram-negative bacteria. Furtheradvantageous regulatory sequences are, for example, present in theGram-positive promoters amy and SPO2, in the yeast or fungal promotersADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plantpromoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Wardet al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33,nos or in the ubiquitin or phaseolin promoter. Advantageous in thiscontext are also inducible promoters, such as the promoters described inEP-A-0 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992:397-404(Gatz et al., tetracycline-inducible), EP-A-0 335 528 (abscisicacid-inducible) or WO 93/21334 (ethanol- or cyclohexenol-inducible).Further suitable plant promoters are the cytosolic FBPase promoter orthe ST-LSI promoter of potato (Stockhaus et al., EMBO J. 8, 1989, 2445),the Glycine max phosphoribosylpyrophosphate amidotransferase promoter(Genbank Accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676. Especially advantageous promoters are promoters whichmake possible the expression in tissues which are involved in thebiosynthesis of fatty acids. Very especially advantageous areseed-specific promoters, such as the USP promoter as described, but alsoother promoters such as the LeB4, DC3, phaseolin or napin promoter.Further especially advantageous promoters are seed-specific promoterswhich can be used for monocotyledonous or dicotyledonous plants andwhich are described in U.S. Pat. No. 5,608,152 (oilseed rape napinpromoter), WO 98/45461 (Arabidopsis oleosin promoter), U.S. Pat. No.5,504,200 (Phaseolus vulgaris phaseolin promoter), WO 91/13980 (BrassicaBce4 promoter), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4promoter from a legume), these promoters being suitable for dicots.Examples of promoters which are suitable for monocots are the barleyIpt-2 or Ipt-1 promoter (WO 95/15389 and WO 95/23230), the barleyhordein promoter and other suitable promoters described in WO 99/16890.

In principle, it is possible to use all natural promoters together withtheir regulatory sequences, such as those mentioned above, for the novelprocess. It is also possible and advantageous to use syntheticpromoters, either in addition or alone, in particular when they mediateseed-specific expression, such as those described in WO 99/16890.

In order to achieve a particularly high PUFA content, especially intransgenic plants, the PUFA biosynthesis genes should advantageously beexpressed in oil crops in a seed-specific manner. To this end,seed-specific promoters can be used, or those promoters which are activein the embryo and/or in the endosperm. In principle, seed-specificpromoters can be isolated both from dicotyledonous and frommonocotyledonous plants. Advantageous preferred promoters are listedhereinbelow: USP (=unknown seed protein) and vicilin (Vicia faba)[Bäumlein et al., Mol. Gen. Genet., 1991, 225(3)], napin (oilseed rape)[U.S. Pat. No. 5,608,152], acyl carrier protein (oilseed rape) [U.S.Pat. No. 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [U.S. Pat. No.5,504,200], Bce4 [WO 91/13980], legumes B4 (LegB4 promoter) [Bäumlein etal., Plant J., 2,2, 1992], Lpt2 and Ipt1 (barley) [WO 95/15389 and WO95/23230], seed-specific promoters from rice, maize and wheat [WO99/16890], Amy32b, Amy 6-6 and aleurain [U.S. Pat. No. 5,677,474], Bce4(oilseed rape) [U.S. Pat. No. 5,530,149], glycinin (soybean) [EP 571741], phosphoenol pyruvate carboxylase (soybean) [JP 06/62870], ADR12-2(soybean) [WO 98/08962], isocitrate lyase (oilseed rape) [U.S. Pat. No.5,689,040] or α-amylase (barley) [EP 781 849].

Plant gene expression can also be facilitated via a chemically induciblepromoter (see review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol.Biol., 48:89-108). Chemically inducible promoters are particularlysuitable when it is desired that gene expression should take place in atime-specific manner. Examples of such promoters are asalicylic-acid-inducible promoter (WO 95/19443), atetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404)and an ethanol-inducible promoter.

To ensure the stable integration of the biosynthesis genes into thetransgenic plant over a plurality of generations, each of the nucleicacids which code for lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferaseand/or lecithin cholesterol acyltransferase, the advantageous acyl-CoA:lysophospholipid acyltransferase, Δ-4-desaturase, desaturase,Δ-6-desaturase, Δ-8-desaturase and/or Δ-5-elongase, Δ-6-elongase and/orΔ-9-elongase and which are used in the process should be expressed underthe control of a separate promoter, preferably a promoter which differsfrom the other promoters, since repeating sequence motifs can lead toinstability of the T-DNA, or to recombination events. In this context,the expression cassette is advantageously constructed in such a way thata promoter is followed by a suitable cleavage site, advantageously in apolylinker, for insertion of the nucleic acid to be expressed and, ifappropriate, a terminator is positioned behind the polylinker. Thissequence is repeated several times, preferably three, four or fivetimes, so that up to five genes can be combined in one construct andintroduced into the transgenic plant in order to be expressed.Advantageously, the sequence is repeated up to three times. To expressthe nucleic acid sequences, the latter are inserted behind the promotervia the suitable cleavage site, for example in the polylinker.Advantageously, each nucleic acid sequence has its own promoter and, ifappropriate, its own terminator. However, it is also possible to inserta plurality of nucleic acid sequences behind a promoter and, ifappropriate, before a terminator. Here, the insertion site, or thesequence, of the inserted nucleic acids in the expression cassette isnot of critical importance, that is to say a nucleic acid sequence canbe inserted at the first or last position in the cassette without itsexpression being substantially influenced thereby. Advantageously,different promoters such as, for example, the USP, LegB4 or DC3promoter, and different terminators can be used in the expressioncassette. However, it is also possible to use only one type of promoterin the cassette. This, however, may lead to undesired recombinationevents.

As described above, the transcription of the genes which have beenintroduced should advantageously be terminated by suitable terminatorsat the 3′ end of the biosynthesis genes which have been introduced(behind the stop codon). An example of a sequence which can be used inthis context is the OCS1 terminator. As is the case with the promoters,different terminator sequences should be used for each gene.

As described above, the gene construct can also comprise further genesto be introduced into the organisms. It is possible and advantageous tointroduce into the host organisms, and to express therein, regulatorygenes such as genes for inductors, repressors or enzymes which, owing totheir enzyme activity, engage in the regulation of one or more genes ofa biosynthetic pathway. These genes can be of heterologous or ofhomologous origin. Moreover, further biosynthesis genes of the fattyacid or lipid metabolism can advantageously be present in the nucleicacid construct, or gene construct; however, these genes can also bepositioned on one or more further nucleic acid constructs. Biosynthesisgenes of the fatty acid or lipid metabolism which are advantageouslyused are a gene selected from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s), fatty acidhydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme Aoxidase(s), fatty acid desaturase(s), fatty acid acetylenases,lipoxygenase(s), triacylglycerol lipase(s), alleneoxide synthase(s),hydroperoxide lyase(s) or fatty acid elongase(s) or combinationsthereof. Especially advantageous nucleic acid sequences are biosynthesisgenes of the fatty acid or lipid metabolism selected from the groupconsisting of acyl-CoA: lysophospholipid acyltransferase,Δ-4-desaturase, Δ-5-desaturase, Δ-6-desaturase, Δ-8-desaturase,Δ-9-desaturase, Δ-12-desaturase, Δ-5-elongase, Δ-6-elongase orΔ-9-elongase.

In this context, the abovementioned nucleic acids and genes can becloned into expression cassettes of the invention in combination withother elongases and desaturases and used for transforming plants withthe aid of Agrobacterium.

Here, the regulatory sequences or factors can, as described above,preferably have a positive effect on, and thus enhance, the expressionof the genes which have been introduced. Thus, enhancement of theregulatory elements can advantageously take place at the transcriptionallevel by using strong transcription signals such as promoters and/orenhancers. However, an enhanced translation is also possible, forexample by improving the stability of the mRNA. In principle, theexpression cassettes can be used directly for introduction into theplant or else be introduced into a vector.

These advantageous vectors, preferably expression vectors, comprise thenucleic acids which code for lysophosphatidic acid acyltransferases,glycerol-3-phosphate acyltransferases, diacylglycerol acyltransferasesor lecithin cholesterol acyltransferases and which are used in theprocess, or else a nucleic acid construct which comprises the nucleicacid used either alone or in combination with further biosynthesis genesof the fatty acid or lipid metabolism such as theacyl-CoA:lysophospholipid acyltransferases, Δ-4-desaturase,Δ-5-desaturase, Δ-6-desaturase, Δ-8-desaturase, Δ-9-desaturase,Δ-12-desaturase, Δ-5-elongase, Δ-6-elongase and/or Δ-9-elongase. As usedin the present context, the term “vector” refers to a nucleic acidmolecule which is capable of transporting another nucleic acid to whichit is bound. One type of vector is a “plasmid”, a circulardouble-stranded DNA loop into which additional DNA segments can beligated. A further type of vector is a viral vector, it being possiblefor additional DNA segments to be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey have been introduced (for example bacterial vectors with bacterialreplication origin). Other vectors are advantageously integrated intothe genome of a host cell when they are introduced into the host cell,and thus replicate together with the host genome. Moreover, certainvectors can govern the expression of genes with which they are infunctional linkage. These vectors are referred to in the present contextas “expression vectors”. Usually, expression vectors which are suitablefor DNA recombination techniques take the form of plasmids. In thepresent description, “plasmid” and “vector” can be used exchangeablysince the plasmid is the form of vector which is most frequently used.However, the invention is intended to comprise these other forms ofexpression vectors, such as viral vectors, which exert similarfunctions. Furthermore, the term “vector” is also intended to compriseother vectors with which the skilled worker is familiar, such as phages,viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids,phagemids, cosmids, linear or circular DNA.

The recombinant expression vectors advantageously used in the processcomprise the nucleic acids described below or the above-described geneconstruct in a form which is suitable for expressing the nucleic acidsused in a host cell, which means that the recombinant expression vectorscomprise one or more regulatory sequences, selected on the basis of thehost cells to be used for the expression, which regulatory sequence(s)is/are linked functionally with the nucleic acid sequence to beexpressed. In a recombinant expression vector, “linked functionally”means that the nucleotide sequence of interest is bound to theregulatory sequence(s) in such a way that the expression of thenucleotide sequence is possible and they are bound to each other in sucha way that both sequences carry out the predicted function which isascribed to the sequence (for example in an in-vitrotranscription/translation system, or in a host cell if the vector isintroduced into the host cell). The term “regulatory sequence” isintended to comprise promoters, enhancers and other expression controlelements (for example polyadenylation signals). These regulatorysequences are described, for example, in Goeddel: Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990), or see: Gruber and Crosby, in: Methods in Plant MolecularBiology and Biotechnology, CRC Press, Boca Raton, Fla., Ed.: Glick andThompson, Chapter 7, 89-108, including the references cited therein.Regulatory sequences comprise those which govern the constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich govern the direct expression of the nucleotide sequence only inspecific host cells under specific conditions. The skilled worker knowsthat the design of the expression vector can depend on factors such asthe choice of host cell to be transformed, the expression level of thedesired protein and the like.

The recombinant expression vectors used can be designed for theexpression of lysophosphatidic acid acyltransferases,glycerol-3-phosphate acyltransferases, diacylglycerol acyltransferasesor lecithin cholesterol acyltransferases, acyl-CoA: lysophospholipidacyltransferases, desaturases and elongases in prokaryotic or eukaryoticcells. This is advantageous since intermediate steps of the vectorconstruction are frequently carried out in microorganisms for the sakeof simplicity. For example, lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase,lecithin cholesterol acyltransferase, acyl-CoA: lysophospholipidacyltransferase, desaturase and/or elongase genes can be expressed inbacterial cells, insect cells (using Baculovirus expression vectors),yeast and other fungal cells (see Romanos, M. A., et al. (1992) “Foreigngene expression in yeast: a review”, Yeast 8:423-488; van den Hondel,C.A.M.J.J., et al. (1991) “Heterologous gene expression in filamentousfungi”, in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van den Hondel,C.A.M.J.J., & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge UniversityPress: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology.1, 3:239-251), ciliates of the types: Holotrichia, Peritrichia,Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma,Platyophrya, Potomacus, Desaturaseudocohnilembus, Euplotes,Engelmaniella and Stylonychia, in particular of the genus Stylonychialemnae, using vectors in a transformation method as described in WO98/01572 and, preferably, in cells of multi-celled plants (see Schmidt,R. and Willmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.: 583-586; Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., Chapter 6/7, pp. 71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein)).Suitable host cells are furthermore discussed in Goeddel, GeneExpression Technology Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). As an alternative, the recombinant expressionvector can be transcribed and translated in vitro, for example usingT7-promoter regulatory sequences and T7-polymerase.

In most cases, the expression of proteins in prokaryotes involves theuse of vectors comprising constitutive or inducible promoters whichgovern the expression of fusion or nonfusion proteins. Typical fusionexpression vectors are, inter alia, pGEX (Pharmacia Biotech Inc; Smith,D. B., and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), whereglutathione S-transferase (GST), maltose-E binding protein and proteinA, respectively, is fused with the recombinant target protein.

Examples of suitable inducible nonfusion E. coli expression vectors are,inter alia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). The target geneexpression from the pTrc vector is based on the transcription from ahybrid trp-lac fusion promoter by the host RNA polymerase. The targetgene expression from the vector pET 11d is based on the transcription ofa T7-gn10-lac fusion promoter, which is mediated by a viral RNApolymerase (T7 gn1), which is coexpressed. This viral polymerase isprovided by the host strains BL21 (DE3) or HMS174 (DE3) from a residentλ-prophage which harbors a T7 gn1 gene under the transcriptional controlof the lacUV 5 promoter.

Other vectors which are suitable for prokaryotic organisms are known tothe skilled worker, these vectors are, for example in E. coli pLG338,pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 orpUC19, the M113 mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24,pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCI, in Streptomyces pIJ101,pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, inCorynebacterium pSA77 or pAJ667.

In a further embodiment, the expression vector is a yeast expressionvector. Examples for vectors for expression in the yeast S. cerevisiaecomprise pYeDesaturasec1 (Baldari et al. (1987) Embo J. 6:229-234), pMFa(Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al.(1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and processes for the construction of vectors which aresuitable for use in other fungi, such as the filamentous fungi, comprisethose which are described in detail in: van den Hondel, C.A.M.J.J., &Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of fungi, J. F.Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, orin: More Gene Manipulations in Fungi [J. W. Bennet & L. L. Lasure, Ed.,pp. 396-428: Academic Press: San Diego]. Further suitable yeast vectorsare, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.

As an alternative, the lysophosphatidic acid acyltransferases,glycerol-3-phosphate acyltransferases, diacylglycerol acyltransferases,lecithin cholesterol acyltransferases, acyl-CoA: lysophospholipidacyltransferases, desaturases and/or elongases can be expressed ininsect cells using Baculovirus expression vectors. Baculovirus vectorswhich are available for the expression of proteins in cultured insectcells (for example Sf9 cells) comprise the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

The abovementioned vectors offer only a small overview of suitablevectors which are possible. Further plasmids are known to the skilledworker and are described, for example, in: Cloning Vectors (Ed. Pouwels,P. H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444904018). For further suitable expression systems for prokaryotic andeukaryotic cells, see the Chapters 16 and 17 in Sambrook, J., Fritsch,E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2ndedition, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In a further embodiment of the process, the lysophosphatidic acidacyltransferases, glycerol-3-phosphate acyltransferases, diacylglycerolacyltransferases, lecithin cholesterol acyltransferases, acyl-CoA:lysophospholipid acyltransferases, desaturases and/or elongases can beexpressed in single-celled plant cells (such as algae), see Falciatoreet al., 1999, Marine Biotechnology 1 (3):239-251 and references citedtherein, and in plant cells from higher plants (for examplespermatophytes such as arable crops). Examples of plant expressionvectors comprise those which are described in detail in: Becker, D.,Kemper, E., Schell, J., and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acids Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu,Academic Press, 1993, pp. 15-38.

A plant expression cassette preferably comprises regulatory sequenceswhich are capable of governing the expression of genes in plant cellsand which are linked functionally so that each sequence can fulfill itsfunction, such as transcriptional termination, for examplepolyadenylation signals. Preferred polyadenylation signals are thosewhich are derived from Agrobacterium tumefaciens T-DNA, such as gene 3of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.),which is known as octopine synthase, or functional equivalents thereof,but all other terminators which are functionally active in plants arealso suitable.

Since plant gene expression is very often not limited to transcriptionallevels, a plant expression cassette preferably comprises other sequenceswhich are linked functionally, such as translation enhancers, forexample the overdrive sequence, which comprises the tobacco mosaic virus5′-untranslated leader sequence, which increases the protein/RNA ratio(Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).

As described above, plant gene expression must be linked functionallywith a suitable promoter which triggers gene expression with the correcttiming or in a cell- or tissue-specific manner. Utilizable promoters areconstitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), suchas those which are derived from plant viruses, such as 35S CAMV (Francket al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S. Pat. No.5,352,605 and WO 84/02913), or plant promoters, such as the promoter ofthe small rubisco subunit, which is described in U.S. Pat. No.4,962,028.

Other preferred sequences for use in functional linkage in plant geneexpression cassettes are targeting sequences, which are required forsteering the gene product into its corresponding cell compartment (see areview in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 andreferences cited therein), for example into the vacuole, into thenucleus, all types of plastids, such as amyloplasts, chloroplasts,chromoplasts, the extracellular space, the mitochondria, the endoplasmicreticulum, elaioplasts, peroxisomes and other compartments of plantcells.

As described above, plant gene expression can also be facilitated via achemically inducible promoter (see review in Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108). Chemically inducible promotersare particularly suitable when it is desired that the gene expressiontakes place in a time-specific manner. Examples of such promoters are asalicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz et al. (1992) Plant J. 2, 397-404) and anethanol-inducible promoter.

Promoters which respond to biotic or abiotic stress conditions are alsosuitable, for example the pathogen-induced PRP1 gene promoter (Ward etal., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible tomatohsp80 promoter (U.S. Pat. No. 5,187,267), the chill-inducible potatoalpha-amylase promoter (WO 96/12814) or the wound-inducible pinIIpromoter (EP-A-0 375 091).

Especially preferred are those promoters which bring about the geneexpression in tissues and organs in which the biosynthesis of fattyacids, lipids and oils takes place, in seed cells, such as cells of theendosperm and of the developing embryo. Suitable promoters are theoilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Viciafaba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225(3):459-67), the Arabidopsis oleosin promoter (WO 98/45461), thePhaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), theBrassica Bce4 promoter (WO 91/13980) or the legumine B4 promoter (LeB4;Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), and promoters whichbring about the seed-specific expression in monocotyledonous plants suchas maize, barley, wheat, rye, rice and the like. Suitable noteworthypromoters are the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO95/23230) or the promoters from the barley hordein gene, the riceglutelin gene, the rice oryzin gene, the rice prolamine gene, the wheatgliadine gene, the wheat glutelin gene, the maize zeine gene, the oatglutelin gene, the sorghum kasirin gene or the rye secalin gene, whichare described in WO 99/16890.

In particular, it may be desired to bring about the multiparallelexpression of the lysophosphatidic acid acyltransferases,glycerol-3-phosphate acyltransferases, diacylglycerol acyltransferasesor lecithin cholesterol acyltransferases used in the process alone or incombination with acyl-CoA: lysophospholipid acyltransferases,desaturases and/or elongases. Such expression cassettes can beintroduced via the simultaneous transformation of a plurality ofindividual expression constructs or, preferably, by combining aplurality of expression cassettes on one construct. Also, a plurality ofvectors can be transformed with in each case a plurality of expressioncassettes and then transferred onto the host cell.

Promoters which are likewise especially suitable are those which bringabout plastid-specific expression, since plastids constitute thecompartment in which the precursors and some end products of lipidbiosynthesis are synthesized. Suitable promoters, such as the viral RNApolymerase promoter, are described in WO 95/16783 and WO 97/06250, andthe clpP promoter from Arabidopsis, described in WO 99/46394.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction, asused in the present context, are intended to comprise a multiplicity ofmethods known in the prior art for the introduction of foreign nucleicacid (for example DNA) into a host cell, including calcium phosphate orcalcium chloride coprecipitation, DEAE-dextran-mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory textbookssuch as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

Host cells which are suitable in principle for taking up the nucleicacid according to the invention, the gene product according to theinvention or the vector according to the invention are all prokaryoticor eukaryotic organisms. The host organisms which are advantageouslyused are microorganisms such as fungi or yeasts, or plant cells,preferably plants or parts thereof. Fungi, yeasts or plants arepreferably used, especially preferably plants, very especiallypreferably plants such as oil crop plants, which are high in lipidcompounds, such as oilseed rape, evening primrose, hemp, thistle,peanut, canola, linseed, soybean, safflower, sunflower, borage, orplants such as maize, wheat, rye, oats, triticale, rice, barley, cotton,cassava, pepper, Tagetes, Solanaceae plants such as potato, tobacco,eggplant and tomato, Vicia species, pea, alfalfa, bushy plants (coffee,cacao, tea), Salix species, trees (oil palm, coconut), and perennialgrasses and fodder crops. Especially preferred plants according to theinvention are oil crop plants such as soybean, peanut, oilseed rape,canola, linseed, hemp, evening primrose, sunflower, safflower, trees(oil palm, coconut).

The invention furthermore relates to isolated nucleic acid sequences asdescribed above coding for polypeptides having lysophosphatidic acidacyltransferase activity, glycerol-3-phosphate acyltransferase activity,diacylglycerol acyltransferase activity or lecithin cholesterolacyltransferase activity, where the lysophosphatidic acidacyltransferases, glycerol-3-phosphate acyltransferases, diacylglycerolacyltransferases or lecithin cholesterol acyltransferases encoded by thenucleic acid sequences specifically convert C₁₈-, C₂₀-, C₂₂- orC₂₄-fatty acids with at least one double bonds in the fatty acidmolecule.

Advantageous isolated nucleic acid sequences are sequences selected fromthe group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3,

SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20,

-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ    ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:    16, SEQ ID NO: 18 or SEQ ID NO: 20,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:    9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ    ID NO: 18 or SEQ ID NO: 20, which code for polypeptides with the    amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:    8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ    ID NO: 19 or SEQ ID NO: 21 and which have at least 40% homology at    the amino acid level with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8,    SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID    NO: 19 or SEQ ID NO: 21 and have lysophosphatidic acid    acyltransferase activity.

Further advantageous isolated nucleic acid sequences are sequencesselected from the group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 22,    SEQ ID NO: 24 or SEQ ID NO: 26,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 22, SEQ ID NO: 24 or SEQ ID NO: 26,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 22,    SEQ ID NO: 24 or SEQ ID NO: 26, which code for polypeptides with the    amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID    NO: 27 and have at least 40% homology at the amino acid level with    SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 and have    glycerol-3-phosphate acyltransferase activity.

Further advantageous isolated nucleic acid sequences are sequencesselected from the group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 28,    SEQ ID NO: 30 or SEQ ID NO: 32,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 28,    SEQ ID NO: 30 or SEQ ID NO: 32, which code for polypeptides with the    amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID    NO: 33 and have at least 40% homology at the amino acid level with    SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33 and which have    diacylglycerol acyltransferase activity.

Further advantageous isolated nucleic acid sequences are sequencesselected from the group consisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 34    or SEQ ID NO: 36,-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the coding sequence in SEQ ID    NO: 34 or SEQ ID NO: 36,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 34    or SEQ ID NO: 36, which code for polypeptides with the amino acid    sequence shown in SEQ ID NO: 35 or SEQ ID NO: 37 and which have at    least 40% homology at the amino acid level with SEQ ID NO: 35 or SEQ    ID NO: 37 and have lecithin cholesterol acyltransferase activity.

The abovementioned nucleic acids according to the invention are derivedfrom organisms such as animals, ciliates, fungi, plants such as algae ordinoflagellates which are capable of synthesizing PUFAs.

In an advantageous embodiment, the term “nucleic acid (molecule)” asused in the present context additionally comprises the untranslatedsequence at the 3′ and at the 5′ end of the coding gene region: at least500, preferably 200, especially preferably 100 nucleotides of thesequence upstream of the 5′ end of the coding region and at least 100,preferably 50, especially preferably 20 nucleotides of the sequencedownstream of the 3′ end of the coding gene region. An “isolated”nucleic acid molecule is separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. An“isolated” nucleic acid preferably has no sequences which naturallyflank the nucleic acid in the genomic DNA of the organism from which thenucleic acid is derived (for example sequences which are located at the5′ and 3′ ends of the nucleic acid). In various embodiments, theisolated lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase and/or lecithincholesterol acyltransferase molecule can comprise for example fewer thanapproximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule inthe genomic DNA of the cell from which the nucleic acid is derived.

The nucleic acid molecules used in the process, for example a nucleicacid molecule with a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 or of a partthereof can be isolated using molecular-biological standard techniquesand the sequence information provided herein. Also, for example ahomologous sequence or homologous, conserved sequence regions can beidentified at the DNA or amino acid level with the aid of comparativealgorithms. They can be used as hybridization probe together withstandard hybridization techniques (such as, for example, those describedin Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) for isolating further nucleic acid sequenceswhich can be used in the process. Moreover, a nucleic acid moleculecomprising a complete sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 or a part thereof can beisolated by polymerase chain reaction, where oligonucleotide primerswhich are based on this sequence or on parts thereof are used (forexample a nucleic acid molecule comprising the complete sequence or apart thereof can be isolated by polymerase chain reaction usingoligonucleotide primers which have been generated based on this samesequence). For example, mRNA can be isolated from cells (for example bymeans of the guanidinium thiocyanate extraction method of Chirgwin etal. (1979) Biochemistry 18:5294-5299) and cDNA by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, availablefrom Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for the amplification by means of polymerasechain reaction can be generated based on one of the sequences shown inSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 orSEQ ID NO: 36 or with the aid of the amino acid sequences detailed inSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35 or SEQ ID NO: 37. A nucleic acid according to theinvention can be amplified by standard PCR amplification techniquesusing cDNA or, alternatively, genomic DNA as template and suitableoligonucleotide primers. The nucleic acid amplified thus can be clonedinto a suitable vector and characterized by means of DNA sequenceanalysis. Oligonucleotides which correspond to a desaturase nucleotidesequence can be generated by standard synthetic methods, for exampleusing an automatic DNA synthesizer.

Homologs of the lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase orlecithin cholesterol acyltransferase nucleic acid sequences used withthe sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34 or SEQ ID NO: 36 means, for example, allelic variants with at leastapproximately 40 to 60%, preferably at least approximately from 60 to70%, more preferably at least approximately from 70 to 80%, 80 to 90% or90 to 95% and even more preferably at least approximately 95%, 96%, 97%,98%, 99% or more homology with a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 orits homologs, derivatives or analogs or parts thereof. Furthermore,isolated nucleic acid molecules of a nucleotide sequence which hybridizewith one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 or with a partthereof, for example hybridized under stringent conditions. Allelicvariants comprise in particular functional variants which can beobtained by deletion, insertion or substitution of nucleotides from/intothe sequence detailed in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO:6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34 or SEQ ID NO: 36 it being intended, however, that theenzyme activity of the resulting proteins which are synthesized isadvantageously retained for the insertion of one or more genes. Proteinswhich retain the enzymatic activity of lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase, i.e. whoseactivity is essentially not reduced, means proteins with at least 10%,preferably 20%, especially preferably 30%, very especially preferably40% of the original enzyme activity in comparison with the proteinencoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34 or SEQ ID NO: 36.

Homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34 or SEQ ID NO: 36 mean for example also bacterial, fungal and planthomologs, truncated sequences, single-stranded DNA or RNA of the codingand noncoding DNA sequence.

Homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34 or SEQ ID NO: 36 also mean derivatives such as, for example, promotervariants. The promoters upstream of the nucleotide sequences detailedcan be modified by one or more nucleotide exchanges, by insertion(s)and/or deletion(s) without the functionality or activity of thepromoters being adversely affected, however. It is furthermore possiblethat the modification of the promoter sequence enhances their activityor that they are replaced entirely by more active promoters, includingthose from heterologous organisms.

The abovementioned nucleic acids and protein molecules withlysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase activity which are involved in the metabolism of lipidsand fatty acids, PUFA cofactors and enzymes or in the transport oflipophilic compounds across membranes are used in the process accordingto the invention for the modulation of the production of PUFAs intransgenic organisms, advantageously in plants, such as maize, wheat,rye, oats, triticale, rice, barley, soybean, peanut, cotton, Linumspecies such as linseed or flax, Brassica species such as oilseed rape,canola and turnip rape, pepper, sunflower, borage, evening primrose andTagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato,Vicia species, pea, cassava, alfalfa, bushy plants (coffee, cacao, tea),Salix species, trees (oil palm, coconut) and perennial grasses andfodder crops, either directly (for example when the overexpression oroptimization of a fatty acid biosynthesis protein has a direct effect onthe yield, production and/or production efficiency of the fatty acidfrom modified organisms) and/or can have an indirect effect whichnevertheless leads to an enhanced yield, production and/or productionefficiency of the PUFAs or a reduction of undesired compounds (forexample when the modulation of the metabolism of lipids and fatty acids,cofactors and enzymes leads to modifications of the yield, productionand/or production efficiency or the composition of the desired compoundswithin the cells, which, in turn, can affect the production of one ormore fatty acids).

The combination of various precursor molecules and biosynthesis enzymesleads to the production of various fatty acid molecules, which has adecisive effect on lipid composition, since polyunsaturated fatty acids(=PUFAs) are not only incorporated into triacylglycerol but also intomembrane lipids.

Lipid synthesis can be divided into two sections: the synthesis of fattyacids and their binding to sn-glycerol-3-phosphate, and the addition ormodification of a polar head group. Usual lipids which are used inmembranes comprise phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACPby acetyl transacylase. After a condensation reaction, these two productmolecules together form acetoacetyl-ACP, which is converted via a seriesof condensation, reduction and dehydratization reactions so that asaturated fatty acid molecule with the desired chain length is obtained.The production of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (regarding the fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., pp. 612-636 and referencescited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes.Thieme: Stuttgart, N.Y., and the references therein, and Magnuson, K.,et al. (1993) Microbiological Reviews 57:522-542 and the referencestherein). To undergo the further elongation steps, the resultingphospholipid-bound fatty acids must then be returned from thephospholipids to the fatty acid CoA ester pool. This is made possible byacyl-CoA:lysophospholipid acyltransferases. Moreover, these enzymes arecapable of transferring the elongated fatty acids from the CoA estersback to the phospholipids. If appropriate, this reaction sequence can befollowed repeatedly.

Examples of precursors for the biosynthesis of PUFAs are oleic acid,linoleic acid and linolenic acid. These C₁₈-carbon fatty acids must beelongated to C₂₀ and C₂₂ in order to obtain fatty acids of the eicosaand docosa chain type. With the aid of the lysophosphatidic acidacyltransferases, glycerol-3-phosphate acyltransferases, diacylglycerolacyltransferases, lecithin cholesterol acyltransferases used in theprocess, advantageously in combination with acyl-CoA: lysophospholipidacyltransferases, desaturases such as Δ-4-, Δ-6- and Δ-8-desaturasesand/or Δ-5-, elongases, arachidonic acid, eicosapentaenoic acid,docosapentaenoic acid or docosahexaenoic acid and various otherlong-chain PUFAs can be obtained, extracted and employed in variousapplications regarding foodstuffs, feedstuffs, cosmetics orpharmaceuticals. Preferably, C₁₈-, C₂₀-, C₂₂- and/or C₂₄-fatty acidswith at least two, advantageously at least three, four, five or six,double bonds in the fatty acid molecule can be prepared using theabovementioned enzymes, to give preferably C₂₀-, C₂₂- and/or C₂₄-fattyacids with advantageously three, four or five double bonds in the fattyacid molecule. Desaturation may take place before or after elongation ofthe fatty acid in question. This is why the products of the desaturaseactivities and the further desaturation and elongation steps which arepossible result in preferred PUFAs with a higher degree of desaturation,including a further elongation from C₂₀- to C₂₂-fatty acids, to fattyacids such as γ-linolenic acid, dihomo-γ-linolenic acid, arachidonicacid, stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.Substrates of the lysophosphatidic acyltransferases,glycerol-3-phosphate acyltransferases, diacylglycerol acyltransferasesor lecithin cholesterol acyltransferases in the process according to theinvention are C₁₈-, C₂₀- or C₂₂-fatty acids such as, for example,linoleic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenicacid, eicosatetraenoic acid or stearidonic acid. Preferred substratesare linoleic acid, γ-linolenic acid and/or α-linolenic acid,dihomo-γ-linolenic acid, arachidonic acid, eicosatetraenoic acid oreicosapentaenoic acid. The C₁₈-, C₂₀- or C₂₂-fatty acids with at leasttwo double bonds in the fatty acid are obtained in the process accordingto the invention in the form of the free fatty acid or in the form oftheir esters, for example in the form of their glycerides.

The term “glyceride” is understood as meaning a glycerol esterified withone, two or three carboxyl radicals (mono-, di- or triglyceride).“Glyceride” is also understood as meaning a mixture of variousglycerides. The glyceride or glyceride mixture may comprise furtheradditions, for example free fatty acids, antioxidants, proteins,carbohydrates, vitamins and/or other substances.

For the purposes of the process of the invention, a “glyceride” isfurthermore understood as meaning glycerol derivatives. In addition tothe above-described fatty acid glycerides, these also includeglycerophospholipids and glyceroglycolipids. Preferred examples whichmay be mentioned in this context are the glycerophospholipids such aslecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol,phosphatidylserine and alkylacylglycerophospholipids.

Furthermore, fatty acids must subsequently be translocated to variousmodification sites and incorporated into the triacylglycerol storagelipid. A further important step in lipid synthesis is the transfer offatty acids to the polar head groups, for example by glycerol fatty acidacyltransferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).

For publications on plant fatty acid biosynthesis and on thedesaturation, the lipid metabolism and the membrane transport of lipidiccompounds, on beta-oxidation, fatty acid modification and cofactors,triacylglycerol storage and triacylglycerol assembly, including thereferences therein, see the following papers: Kinney, 1997, GeneticEngineering, Ed.: J K Setlow, 19:149-166; Ohlrogge and Browse, 1995,Plant Cell 7:957-970; Shanklin and Cahoon, 1998, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 49:611-641; Voelker, 1996, GeneticEngineering, Ed.: J K Setlow, 18:111-13; Gerhardt, 1992, Prog. Lipid R.31:397-417; Gühnemann-Schäfer & Kindl, 1995, Biochim. Biophys Acta1256:181-186; Kunau et al., 1995, Prog. Lipid Res. 34:267-342; Stymne etal., 1993, in: Biochemistry and Molecular Biology of Membrane andStorage Lipids of Plants, Ed.: Murata and Somerville, Rockville,American Society of Plant Physiologists, 150-158, Murphy & Ross 1998,Plant Journal. 13(1):1-16.

The PUFAs produced in the process comprise a group of molecules whichhigher animals are no longer capable of synthesizing and must thereforetake up, or which higher animals are no longer capable of synthesizingthemselves in sufficient quantity and must therefore take up additionalquantities, although they are synthesized readily by other organismssuch as bacteria; for example, cats are no longer capable ofsynthesizing arachidonic acid.

The term “lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase” comprises for the purposes of the invention proteinswhich participate in the biosynthesis of fatty acids and their homologs,derivatives and analogs. Phospholipids for the purposes of the inventionare understood as meaning phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol and/or phosphatidylinositol,advantageously phosphatidylcholine. The terms lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase nucleic acidsequence(s) comprise nucleic acid sequences which code for alysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase and part of which may be a coding region and likewisecorresponding 5′ and 3′ untranslated sequence regions. The termsproduction or productivity are known in the art and encompass theconcentration of the fermentation product (compounds of the formula I)which is formed within a specific period of time and in a specificfermentation volume (for example kg of product per hour per liter). Theterm production efficiency comprises the time required for obtaining aspecific production quantity (for example the time required by the cellto establish a certain throughput rate of a fine chemical). The termyield or product/carbon yield is known in the art and comprises theefficiency of the conversion of the carbon source into the product (i.e.the fine chemical). This is usually expressed for example as kg ofproduct per kg of carbon source. By increasing the yield or productionof the compound, the amount of the molecules obtained of this compound,or of the suitable molecules of this compound obtained in a specificculture quantity over a specified period of time is increased. The termsbiosynthesis or biosynthetic pathway are known in the art and comprisethe synthesis of a compound, preferably of an organic compound, by acell from intermediates, for example in a multi-step and stronglyregulated process. The terms catabolism or catabolic pathway are knownin the art and comprise the cleavage of a compound, preferably of anorganic compound, by a cell to give catabolites (in more general terms,smaller or less complex molecules), for example in a multi-step andstrongly regulated process. The term metabolism is known in the art andcomprises the totality of the biochemical reactions which take place inan organism. The metabolism of a certain compound (for example themetabolism of a fatty acid) thus comprises the totality of thebiosynthetic pathways, modification pathways and catabolic pathways ofthis compound in the cell which relate to this compound.

In a further embodiment, derivatives of the nucleic acid moleculeaccording to the invention represented in SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 code for proteinswith at least 40%, advantageously from approximately 50 to 60%,preferably at least from approximately 60 to 70% and more preferably atleast from approximately 70 to 80%, 80 to 90%, 90 to 95% and mostpreferably at least approximately 96%, 97%, 98%, 99% or more homology(=identity) with a complete amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 orSEQ ID NO: 37. The homology was calculated over the entire amino acid ornucleic acid sequence region. The program PileUp (J. Mol. Evolution.,25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or theprograms Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48;443-453 (1970) and Smith and Waterman (Adv. Appl. Math. 2; 482-489(1981)], which are part of the GCG software packet [Genetics ComputerGroup, 575 Science Drive, Madison, Wis., USA 53711 (1991)], were usedfor the sequence alignment. The sequence homology values which areindicated above as percentages were determined over the entire sequenceregion using the program BestFit and the following settings: Gap Weight:8, Length Weight: 2.

Moreover, the invention comprises nucleic acid molecules which differfrom one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 (and partsthereof) owing to the degeneracy of the genetic code and which thus codefor the same lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase as those encoded by the nucleotide sequences shown inSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 orSEQ ID NO: 36.

In addition to the lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase orlecithin cholesterol acyltransferase nucleotide sequences shown in SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO:36, the skilled worker will recognize that DNA sequence polymorphismswhich lead to changes in the amino acid sequences of thelysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase may exist within a population. These geneticpolymorphisms in the lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase orlecithin cholesterol acyltransferase gene may exist between individualswithin a population owing to natural variation. These natural variantsusually bring about a variance of 1 to 5% in the nucleotide sequence ofthe lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase gene. Each and every one of these nucleotide variationsand resulting amino acid polymorphisms in the lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase which are theresult of natural variation and do not modify the functional activity ofare to be encompassed by the invention.

Owing to their homology to the lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase orlecithin cholesterol acyltransferase nucleic acids disclosed here,nucleic acid molecules which are advantageous for the process accordingto the invention can be isolated following standard hybridizationtechniques under stringent hybridization conditions, using the sequencesor part thereof as hybridization probe. In this context it is possible,for example, to use isolated nucleic acid molecules which are at least15 nucleotides in length and which hybridize under stringent conditionswith the nucleic acid molecules which comprise a nucleotide sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 orSEQ ID NO: 36. Nucleic acids with at least 25, 50, 100, 250 or morenucleotides can also be used. The term “hybridizes under stringentconditions” as used in the present context is intended to describehybridization and washing conditions under which nucleotide sequenceswith at least 60% homology to one another usually remain hybridized withone another. Conditions are preferably such that sequences with at leastapproximately 65%, preferably at least approximately 70% and especiallypreferably at least approximately 75% or more homology to one anotherusually remain hybridized with one another. These stringent conditionsare known to the skilled worker and can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred nonlimiting example of stringent hybridization conditions ishybridizations in 6× sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more washing steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ depending on the type of nucleic acidand, for example when organic solvents are present, regardingtemperature and buffer concentration. Under “standard hybridizationconditions”, for example, the temperature is, depending on the type ofnucleic acid, between 42° C. and 58° C. in aqueous buffer with aconcentration of 0.1 to 5×SSC (pH 7.2). If organic solvent, for example50% formamide, is present in the abovementioned buffer, the temperatureunder standard conditions is approximately 42° C. The hybridizationconditions for DNA:DNA hybrids, for example, are preferably 0.1×SSC and20° C. to 45° C., preferably 30° C. to 45° C. The hybridizationconditions for DNA:RNA hybrids are, for example, preferably 0.1×SSC and30° C. to 55° C., preferably 45° C. to 55° C. The abovementionedhybridization temperatures are determined by way of example for anucleic acid with approximately 100 bp (=base pairs) in length and witha G+C content of 50% in the absence of formamide. The skilled workerknows how to determine the required hybridization conditions on thebasis of the abovementioned textbooks or textbooks such as Sambrook etal., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames andHiggins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”,IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991,“Essential Molecular Biology: A Practical Approach”, IRL Press at OxfordUniversity Press, Oxford.

In order to determine the percentage of homology (=identity) of twoamino acid sequences (for example one of the sequences of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35 or SEQ ID NO: 37) or of two nucleic acids (for example SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36),the sequences are written one under the other for an optimal comparison(for example, gaps may be introduced into the sequence of a protein orof a nucleic acid in order to generate an optimal alignment with theother protein or the other nucleic acid). Then, the amino acid residuesor nucleotides at the corresponding amino acid positions or nucleotidepositions are compared. If a position in a sequence is occupied by thesame amino acid residue or the same nucleotide as the correspondingposition in the other sequence, then the molecules are homologous atthis position (i.e. amino acid or nucleic acid “homology” as used in thepresent context corresponds to amino acid or nucleic acid “identity”).The percentage of homology between the two sequences is a function ofthe number of identical positions which the sequences share (i.e. %homology=number of identical positions/total number of positions×100).The terms homology and identity are therefore to be considered assynonymous. The programs and algorithms used are described above.

An isolated nucleic acid molecule which codes for a lysophosphatidicacid acyltransferase, glycerol-3-phosphate acyltransferase,diacylglycerol acyltransferase or lecithin cholesterol acyltransferasewhich is homologous to a protein sequence of SEQ ID NO: 2, SEQ ID NO: 5,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 orSEQ ID NO: 37 can be generated by introducing one or more nucleotidesubstitutions, additions or deletions into a nucleotide sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO:36 so that one or more amino acid substitutions, additions or deletionsare introduced into the protein which is encoded. Mutations in one ofthe sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34 or SEQ ID NO: 36 can be introduced by standard techniques such assite-specific mutagenesis and PCR-mediated mutagenesis. It is preferredto generate conservative amino acid substitutions in one or more of thepredicted nonessential amino acid residues. In a “conservative aminoacid substitution”, the amino acid residue is replaced by an amino acidresidue with a similar side chain. Families of amino acid residues withsimilar side chains have been defined in the art. These familiescomprise amino acids with basic side chains (for example lysine,arginine, histidine), acidic side chains (for example aspartic acid,glutamic acid), uncharged polar side chains (for example glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), unpolarside chains (for example alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (forexample threonine, valine, isoleucine) and aromatic side chains (forexample tyrosine, phenylalanine, tryptophan, histidine). A predictednonessential amino acid residue in a lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase is thuspreferably replaced by another amino acid residue from the same familyof side chains. In another embodiment, the mutations can, alternatively,be introduced randomly over all or part of the sequence coding forlysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase, for example by saturation mutagenesis, and theresulting mutants can be screened by the herein-describedlysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase activity in order to identify mutants which haveretained the lysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase activity. Following the mutagenesis of one of thesequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34 or SEQ ID NO: 36, the protein which is encoded can be expressedrecombinantly, and the activity of the protein can be determined, forexample using the tests described in the present text.

The present invention is illustrated in greater detail by the exampleswhich follow, which are not to be construed as limiting. The content ofall of the references, patent applications, patents and published patentapplications cited in the present patent application is herewithincorporated by reference.

EXAMPLES Example 1 General Methods A) General Cloning Methods:

Cloning methods such as, for example, restriction cleavages, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linking of DNA fragments,transformation of Escherichia coli and yeast cells, cultivation ofbacteria and sequence analysis of recombinant DNA were carried out asdescribed in Sambrook et al. (1989) (Cold Spring Harbor LaboratoryPress: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994)“Methods in Yeast Genetics” (Cold Spring Harbor Laboratory Press: ISBN0-87969-451-3).

b) Chemicals

Unless stated otherwise in the text, the chemicals used were obtained inanalytical-grade quality from Fluka (Neu-Ulm, Germany), Merck(Darmstadt, Germany), Roth (Karlsruhe, Germany), Serva (Heidelberg,Germany) and Sigma (Deisenhofen, Germany). Solutions were prepared usingpurified, pyrogen-free water, referred to as H₂O hereinbelow, from aMilli-Q Water System water purification system (Millipore, Eschborn,Germany). Restriction endonucleases, DNA-modifying enzymes andmolecular-biological kits were obtained from AGS (Heidelberg, Germany),Amersham (Brunswick, Germany), Biometra (Göttingen, Germany), Boehringer(Mannheim, Germany), Genomed (Bad Oeynhausen, Germany), New EnglandBiolabs (Schwalbach/Taunus, Germany), Novagen (Madison, Wis., USA),Perkin-Elmer (Weiterstadt, Germany), Pharmacia (Freiburg, Germany),Qiagen (Hilden, Germany) and Stratagene (Amsterdam, the Netherlands).Unless stated otherwise, they were used according to the manufacturer'sinstructions.

c) Cloning and Expression of Desaturases and Elongases

The Escherichia coli strain XL1 Blue MRF′ kan (Stratagene) was used forsubcloning Δ-6-desaturase from Physcomitrella patens. This gene wasfunctionally expressed using the Saccharomyces cerevisiae strain INVSc 1(Invitrogen Co.). E. coli was cultured in Luria-Bertani broth (LB,Duchefa, Haarlem, the Netherlands) at 37° C. If necessary, ampicillin(100 mg/liter) was added and 1.5% (w/v) agar was added for solid LBmedia. S. cerevisiae was cultured at 30° C. either in YPG medium or incomplete minimal medium without uracil (CMdum; see in: Ausubel, F. M.,Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,Struhl, K., Albright, L. B., Coen, D. M., and Varki, A. (1995) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York) with either2% (w/v) raffinose or glucose. For solid media, 2% (w/v) Bacto™-Agar(Difco) were added. The plasmids used for cloning and expression arepUC18 (Pharmacia) and pYES2 (Invitrogen Co.).

d) Cloning and Expression of PUFA-Specific Desaturases and Elongases

For expression in plants, cDNA clones of SEQ ID NO: 46 (Physcomitrellapatens Δ-6-desaturase), 48 (Physcomitrella patens Δ-6-elongase) or 50(Phaeodactylum tricornutum Δ-5-desaturase) were modified so as for onlythe coding region to be amplified by means of polymerase chain reactionwith the aid of two oligonucleotides. Care was taken here to observe aconsensus sequence upstream of the start codon, for efficienttranslation. To this end, either the ATA or the AAA base sequence waschosen and inserted into the sequence upstream of the ATG [Kozak, M.(1986) Point mutations define a sequence flanking the AUG initiatorcodon that modulates translation by eukaryotic ribosomes, Cell 44,283-2929]. In addition, a restriction cleavage site was introducedupstream of this consensus triplet, which must be compatible with thecleavage site of the target vector into which the fragment is to becloned and with the aid of which gene expression is to be carried out inmicroorganisms or plants.

The PCR reaction was carried out in a thermocycler (Biometra), usingplasmid DNA as template and Pfu DNA polymerase (Stratagene) and thefollowing temperature program: 3 min at 96° C., followed by 30 cycles of30 s at 96° C., 30 s at 55° C. and 2 min at 72° C., 1 cycle of 10 min at72° C. and stop at 4° C. The annealing temperature was varied dependingon the oligonucleotides chosen. A synthesis time of about one minute perkilobase pair of DNA has to be taken as starting point. Other parameterswhich influence the PCR, such as, for example, Mg ions, salt, DNApolymerase etc., are familiar to the skilled worker in the field and maybe varied as required.

The correct size of the amplified DNA fragment was confirmed by means ofagarose-TBE gel electrophoresis. The amplified DNA was extracted fromthe gel using the QIAquick gel extraction kit (QIAGEN) and ligated intothe SmaI restriction site of the dephosphorylated pUC18 vector, usingthe Sure Clone Ligations Kit (Pharmacia), resulting in the pUCderivatives. After transformation of E. coli XL1 Blue MRF′ kan a DNAminipreparation [Riggs, M. G., & McLachlan, A. (1986) A simplifiedscreening procedure for large numbers of plasmid mini-preparation.BioTechniques 4, 310-313] of ampicillin-resistant transformants wascarried out, and positive clones were identified by means of BamHIrestriction analysis. The sequence of the cloned PCR product wasconfirmed by means of resequencing using the ABI PRISM Big DyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,Weiterstadt, Germany).

e) Transformation of Agrobacterium

Unless described otherwise, Agrobacterium-mediated plant transformationwas carried out with the aid of an Agrobacterium tumefaciens strain, asby Deblaere et al. (1984, Nucl. Acids Res. 13, 4777-4788).

f) Plant Transformation

Unless described otherwise, Agrobacterium-mediated plant transformationwas carried out using standard transformation and regenerationtechniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant MolecularBiology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, inSect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick,Bernard R., Thompson, John E., Methods in Plant Molecular Biology andBiotechnology, Boca Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).

According thereto, it is possible to transform, for example, oilseedrape by means of cotyledon or hypocotyl transformation (Moloney et al.,Plant Cell 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989)694-701). The use of antibiotics for the selection of agrobacteria andplants depends on the binary vector used for transformation and theAgrobacterium strain. Normally, oilseed rape is selected using kanamycinas selectable plant marker.

The transformation of soybean may be carried out using, for example, atechnique described in EP-A-0 0424 047 (Pioneer Hi-Bred International)or in EP-A-0 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770(University Toledo).

The transformation of plants using particle bombardment, polyethyleneglycol-mediated DNA uptake or via the silicon carbonate fiber techniqueis described, for example, by Freeling and Walbot “The maize handbook”(1993) ISBN 3-540-97826-7, Springer Verlag New York).

Unless described otherwise, Agrobacterium-mediated gene transfer intolinseed (Linum usitatissimum) was carried out by the technique asdescribed in Mlynarova et al. [(1994) Plant Cell Report 13:282-285].

G) Plasmids for Plant Transformation

Binary vectors based on the vectors pBinAR (Höfgen and Willmitzer, PlantScience 66 (1990) 221-230) or pGPTV (Becker et al. 1992, Plant Mol.Biol. 20:1195-1197) were used for plant transformation. The binaryvectors which comprise the nucleic acids to be expressed are constructedby ligating the cDNA in sense orientation into the T-DNA. 5′ of thecDNA, a plant promoter activates cDNA transcription. A polyadenylationsequence is located 3′ of the cDNA. The binary vectors may carrydifferent marker genes such as, for example, the acetolactate synthasegene (AHAS or ALS) [Ott et al., J. Mol. Biol. 1996, 263:359-360] whichimparts a resistance to the imidazolinones or the nptII marker genewhich codes for a kanamycin resistance imparted by neomycinphosphotransferase.

Tissue-specific expression of the nucleic acids can be achieved using atissue-specific promoter. Unless described otherwise, the LeB4 or theUSP promoter or the phaseolin promoter was cloned 5′ of the cDNA.Terminators used were the NOS terminator and the OCS terminator (seeFIG. 1). FIG. 1 depicts a vector map of the vector used for expression,pSUN3CeLPLAT.

It is also possible to use any other seed-specific promoter element suchas, for example, the napin or arcelin promoter (Goossens et al. 1999,Plant Phys. 120(4):1095-1103 and Gerhardt et al. 2000, Biochimica etBiophysica Acta 1490(1-2):87-98).

The CaMV-35S promoter or a v-ATPase C1 promoter can be used forconstitutive expression in the whole plant.

The nucleic acids used in the process which code foracyl-CoA:lysophospholipid acyltransferases; desaturases or elongaseswere cloned into a binary vector one after the other by constructing aplurality of expression cassettes, in order to mimic the metabolicpathway in plants.

Within an expression cassette, the protein to be expressed may betargeted into a cellular compartment by using a signal peptide, forexample for plastids, mitochondria or the endoplasmic reticulum(Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423). The signalpeptide is cloned 5′ of and in-frame with the cDNA in order to achievethe subcellular localization of the fusion protein.

Examples of multiexpression cassettes were disclosed in DE 102 19 203and are given again below.

i.) Promoter-Terminator Cassettes

Expression cassettes consist of at least two functional units such as apromoter and a terminator. Further desired gene sequences such astargeting sequences, coding regions of genes or parts thereof etc. maybe inserted between promoter and terminator. To construct the expressioncassettes, promoters and terminators (USP promoter: Baeumlein et al.,Mol Gen Genet, 1991, 225 (3):459-67); OCS terminator: Gielen et al. EMBOJ. 3 (1984) 835ff.) were isolated with the aid of the polymerase chainreaction and tailor-made with flanking sequences of choice on the basisof synthetic oligonucleotides.

Examples of oligonucleotides which may be used are the following:

USP1 upstream (SEQ ID NO: 75):- CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCC A -USP2 upstream (SEQ ID NO: 76):- CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCC A -USP3 upstream (SEQ ID NO: 77):- CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCC A -USP1 downstream (SEQ ID NO: 78):- AAAACTGCAGGCGGCCGCCCACCGCGGTGGGCTGGCTATGAAGAAAT T -USP2 downstream (SEQ ID NO: 79): - CGCGGATCCGCTGGCTATGAAGAAATT -USP3 downstream (SEQ ID NO: 80):- TCCCCCGGGATCGATGCCGGCAGATCTGCTGGCTATGAAGAAATT -OCS1 upstream (SEQ ID NO: 81):- AAAACTGCAGTCTAGAAGGCCTCCTGCTTTAATGAGATAT -OCS2 upstream (SEQ ID NO: 82):- CGCGGATCCGATATCGGGCCCGCTAGCGTTAACCCTGCTTTAATGAG ATAT -OCS3 upstream (SEQ ID NO: 83): - TCCCCCGGGCCATGGCCTGCTTTAATGAGATAT -OCS1 downstream (SEQ ID NO: 84):- CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTA AATTGA -OCS2 downstream (SEQ ID NO: 85):- CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTA AATTGA -OCS3 downstream (SEQ ID NO: 86):- CCCAAGCTTGGCGCGCCGAGCTCGTCGACGGACAATCAGTAAATTG A -

The methods are known to the skilled worker in the field and are wellknown from the literature.

In a first step, a promoter and a terminator were amplified via PCR. Theterminator was then cloned into a recipient plasmid and, in a secondstep, the promoter was inserted upstream of the terminator. As a result,an expression cassette was cloned into the basic plasmid. The plasmidspUT1, 2 and 3 were thus generated on the basis of the pUC19 plasmid.

The corresponding constructs or plasmids are defined in SEQ ID NO: 52,53 and 54. They comprise the USP promoter and the OCS terminator. Basedon these plasmids, the construct pUT12 was generated by cutting pUT1 bymeans of SalI/ScaI and pUT2 by means of XhoI/ScaI. The fragmentscomprising the expression cassettes were ligated and transformed into E.coli XL1 blue MRF. After isolating ampicillin-resistant colonies, DNAwas prepared and those clones which comprise two expression cassetteswere identified by restriction analysis. The XhoI/SalI ligation ofcompatible ends has eliminated here the two cleavage sites, XhoI andSalI, between the expression cassettes. The resulting plasmid, pUT12, isindicated in SEQ ID NO: 55. Subsequently, pUT12 was cut again by meansof Sal/ScaI and pUT3 was cut by means of XhoI/ScaI. The fragmentscomprising the expression cassettes were ligated and transformed into E.coli XLI blue MRF. After isolation from ampicillin-resistant colonies,DNA was again prepared, and those clones which comprise three expressioncassettes were identified by restriction analysis. In this manner, a setof multiexpression cassettes was produced which can be utilized forinsertion of desired DNA and which is described in table 1 and whichmoreover can incorporate further expression cassettes.

Said cassettes comprise the following elements:

TABLE 1 Cleavage sites Cleavage sites PUC19 upstream of the USP Multipledownstream of the OCS derivative promoter cloning cleavage sitesterminator PUT1 EcoRI/AscI/SacI/XhoI BstXI/NotI/PstI/XbaI/StuISalI/EcoRI/SacI/AscI/ HindIII PUT2 EcoRI/AscI/SacI/XhoIBamHI/EcoRV/ApaI/NheI/HpaI SalI/EcoRI/SacI/AscI/ HindIII PUT3EcoRI/AscI/SacI/XhoI BglII/NaeI/ClaI/SmaI/NcoI SalI/SacI/AscI/HindIIIPUT12 EcoRI/AscI/SacI/XhoI BstXI/NotI/PstI/XbaI/StuISalI/EcoRI/SacI/AscI/ double and HindIII expressionBamHI/EcoRV/ApaI/NheI/HpaI cassette PUT123 EcoRI/AscI/SacI/XhoI 1.BstXI/NotI/PstI/XbaI/StuI SalI/SacI/AscI/HindIII triple and expression2. BamHI/EcoRV/ApaI/NheI/HpaI cassette and 3. BglII/NaeI/ClaI/SmaI/NcoI

Furthermore, further multiexpression cassettes may be generated, asdescribed and as specified in more detail in table 2, with the aid ofthe

-   i) USP promoter or with the aid of the-   ii) 700 base pair 3′ fragment of the LeB4 promoter or with the aid    of the-   iii) DC3 promoter and employed for seed-specific gene expression.

The DC3 promoter is described in Thomas, Plant Cell 1996, 263:359-368and consists merely of the region from −117 to +26, which is why ittherefore constitutes one of the smallest known seed-specific promoters.The expression cassettes may comprise several copies of the samepromoter or else be constructed via three different promoters.

Advantageously used polylinker- or polylinker-terminator-polylinkers canbe found in the sequences SEQ ID NO: 60 to 62.

TABLE 2 Multiple expression cassettes Plasmid name of Cleavage sitesCleavage sites the pUC19 upstream of the Multiple downstream of thederivative particular promoter cloning cleavage sites OCS terminatorpUT1 EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuISalI/EcoRI/Sacl/AscI/ (pUC19 with HindIII USP-OCS1) PDCTEcoRI/AscI/SacI/XhoI (2) BamHI/EcoRV/ApaI/NheI/ SalI/EcoRI/Sacl/AscI/(pUC19 with HpaI HindIII DC3-OCS) PleBT EcoRI/AscI/SacI/XhoI (3)BglII/NaeI/ClaI/SmaI/NcoI SalI/SacI/AscI/HindIII (pUC19 withLeB4(700)-OCS) PUD12 EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuISalI/EcoRI/Sacl/AscI/ (pUC 19 with and HindIII USP-OCS1 and (2)BamHI/EcoRV/ApaI/NheI/ with DC3-OCS) HpaI PUDL123 EcoRI/AscI/SacI/XhoI(1) BstXI/NotI/PstI/XbaI/StuI and SalI/SacI/AscI/HindIII Tripleexpression (2) BamHI/(EcoRV*)/ApaI/NheI/ cassette HpaI and (pUC19 with(3) BglII/NaeI/ClaI/SmaI/NcoI USP/DC3 and LeB4-700) *EcoRV cleavage sitecuts in the 700 base pair fragment of the LeB4 promoter (LeB4-700)

Further promoters for multigene constructs can be generated analogously,in particular by using the

-   a) 2.7 kB fragment of the LeB4 promoter or with the aid of the-   b) phaseolin promoter or with the aid of the-   c) constitutive v-ATPase c1 promoter.

It may be particularly desirable to use further particularly suitablepromoters for constructing seed-specific multiexpression cassettes, suchas, for example, the napin promoter or the arcelin-5 promoter.

Further vectors which can be utilized in plants and which have one ortwo or three promoter-terminator expression cassettes can be found inthe sequences SEQ ID NO: 63 to SEQ ID NO: 68.

-   ii.) Generation of expression constructs which comprise promoter,    terminator and desired gene sequence for the expression of PUFA    genes in plant expression cassettes.

The Δ-6-elongase Pp_PSE1 is first inserted into the first cassette inpUT123 via BstXI and XbaI. Then, the moss Δ-6-desaturase (Pp_des6) isinserted via BamHI/NaeI into the second cassette and, finally, thePhaeodactylum Δ-5-desaturase (Pt_des5) is inserted via BglII/NcoI intothe third cassette (see SEQ ID NO: 56). The triple construct is namedpARA1. Taking into consideration sequence-specific restriction cleavagesites, further expression cassettes, as set out in table 3 and referredto as pARA2, pARA3 and pARA4, may be generated.

TABLE 3 Combinations of desaturases and elongases Gene plasmidΔ-6-Desaturase Δ-5-Desaturase Δ-6-Elongase pARA1 Pp_des6 Pt_des5 Pp_PSE1pARA2 Pt_des6 Pt_des5 Pp_PSE1 pARA3 Pt_des6 Ce_des5 Pp_PSE1 PARA4Ce_des6 Ce_des5 Ce_PSE1

-   des5=PUFA-specific Δ-5-desaturase-   des6=PUFA-specific Δ-6-desaturase-   PSE=PUFA-specific Δ-6-elongase-   Pt_des5=Δ-5-desaturase from Phaeodactylum tricornutum-   Pp_des6 or Pt des6=Δ-6-desaturase from Physcomitrella patens or    Phaeodactylum tricornutum-   Pp=Physcomitrella patens, Pt=Phaeodactylum tricornutum-   Pp_PSE1=Δ-6-elongase from Physcomitrella patens-   Pt_PSE1=Δ-6-elongase from Phaeodactylum tricornutum    Ce_des5=Δ-5-desaturase from Caenorhabditis elegans (Genbank Acc. No.    AF078796)-   Ce_des6=Δ-6-desaturase from Caenorhabditis elegans (Genbank Acc. No.    AF031477, bases 11-1342)-   Ce_PSE1=Δ-6-elongase from Caenorhabditis elegans (Genbank Acc. No.    AF244356, bases 1-867)

Further desaturases or elongase gene sequences may also be inserted intoexpression cassettes of the type described, such as, for example,Genbank Acc. No. AF231981, NM_(—)013402, AF206662, AF268031, AF226273,AF110510 or AF110509.

-   iii.) Transfer of expression cassettes into vectors for the    transformation of Agrobacterium tumefaciens and for the    transformation of plants

The constructs thus generated were inserted into the binary vector pGPTVby means of AscI. For this purpose, the multiple cloning sequence wasextended by an AscI cleavage site. For this purpose, the polylinker wassynthesized de novo in the form of two double-stranded oligonucleotides,with an additional AscI DNA sequence being inserted. The oligonucleotidewas inserted into the pGPTV vector by means of EcoRI and HindIII. Thecloning techniques required are known to the skilled worker and mayreadily be found in the literature as described in example 1.

The nucleic acid sequences for Δ-5-desaturase (SEQ ID NO: 50),Δ-6-desaturase (SEQ ID NO: 46) and Δ-6-elongase (SEQ ID NO: 48), whichwere used for the experiments described below, were the sequences fromPhyscomitrella patens and Phaeodactylum tricornutum. The correspondingamino acid sequences can be found in the sequences SEQ ID NO: 47, SEQ IDNO: 49 and SEQ ID NO: 51. A vector which comprises all of theabovementioned genes is indicated in SEQ ID NO: 56. The correspondingamino acid sequences of the genes can be found in SEQ ID NO: 57, SEQ IDNO: 58 and SEQ ID NO: 59.

Example 2 Cloning and Characterization of the ceLPLATs (SEQ ID NO:38-44) a) Database Search

The ceLPLATs (=acyl-CoA:lysophospholipid acyltransferase fromCaenorhabditis elegans) were identified by sequence comparisons withknown LPA-ATs. The search was restricted to the nematode genome(Caenorhabditis elegans) with the aid of the BLAST-Psi algorithm(Altschul et al., J. Mol. Biol. 1990, 215: 403-410), since this organismsynthesizes LCPUFAs. The probe employed in the sequence comparison wasan LPAAT protein sequence from Mus musculus (MsLPAAT Accession No.NP_(—)061350). LPLAT catalyzes, by a reversible transferase reaction,the ATP-independent synthesis of acyl-CoAs from phospholipids with theaid of CoA as cofactor (Yamashita et al., J. Biol. Chem. 2001, 20:26745-26752). Sequence comparisons enabled two putative ceLPLATsequences to be identified (Accession No. T06E8.1 and F59F4.4). Theidentified sequences are most similar to each other and to MsLPAATs(FIG. 2). The alignment was generated using the Clustal program.

b) Cloning of the CeLPLATs

Primer pairs were synthesized on the basis of the ceLPLAT nucleic acidsequences (table 4) and the corresponding cDNAs were isolated from a C.elegans cDNA library by means of PCR processes. The respective primerpairs were selected so as to carry, apart from the start codon, theyeast consensus sequence for high-efficiency translation (Kozak, Cell1986, 44:283-292). The LPLAT cDNAs were amplified in each case using 2μl of cDNA-library solution as template, 200 μM dNTPs, 2.5 U of“proof-reading” pfu polymerase and 50 μmol of each primer in a totalvolume of 50 μl. The conditions for the PCR were as follows: firstdenaturation at 95° C. for 5 minutes, followed by 30 cycles at 94° C.for 30 seconds, 58° C. for one minute and 72° C. for 2 minutes, and afinal extension step at 72° C. for 10 minutes. The sequence of the LPLATcDNAs was confirmed by DNA sequencing.

TABLE 4  Nucleotide sequences of the PCR primers for cloning CeLPLATsPrimer Nucleotide sequence 5′ T06E8.1f* (SEQ ID NO: 87) 5′ACATAATGGAGAACTTCTGGTCGATCGTC 3′ 3′ T06E8.1r* (SEQ ID NO: 88) 5′TTACTCAGATTTCTTCCCGTCTTT 3′ 5′ F59F4.4f* (SEQ ID NO: 89) 5′ACATAATGACCTTCCTAGCCATATTA 3′ 3′ F59F4.4r* (SEQ ID NO: 90) 5′TCAGATATTCAAATTGGCGGCTTC 3′ *f: forward, r: reverse

Example 3 Analysis of the Effect of the Recombinant Proteins onProduction of the Desired Product a) Possible Preparation Methods

The effect of genetic modification in fungi, algae, ciliates or, asdescribed in the examples hereinabove, on the production of thepolyunsaturated fatty acids in yeasts, or in plants may be determined bygrowing the modified microorganisms or the modified plant under suitableconditions (such as those described above) and studying the mediumand/or the cellular components for increased production of the lipids orfatty acids. These analytical techniques are known to the skilled workerand comprise spectroscopy, thin-layer chromatography, various types ofstaining methods, enzymic and microbiological methods and analyticalchromatography such as high-performance liquid chromatography (see, forexample, Ullmann, Encyclopedia of Industrial Chemistry, vol. A2, pp.89-90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)“Applications of HPLC in Biochemistry” in: Laboratory Techniques inBiochemistry and Molecular Biology, vol. 17; Rehm et al. (1993)Biotechnology, vol. 3, chapter III: “Product recovery and purification”,pp. 469-714, VCH: Weinheim; Belter, P. A., et al. (1988) Bioseparations:downstream processing for Biotechnology, John Wiley and Sons; Kennedy,J. F., and Cabral, J. M. S. (1992) Recovery processes for biologicalMaterials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D.(1988) Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, vol. B3; chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

Apart from the abovementioned methods for detecting fatty acids inyeasts, plant lipids are extracted from plant material as described byCahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22):12935-12940, andBrowse et al. (1986) Analytic Biochemistry 152:141-145. The qualitativeand quantitative analysis of lipids or fatty acids is described inChristie, William W., Advances in Lipid Methodology, Ayr/Scotland: OilyPress (Oily Press Lipid Library; 2); Christie, William W., GasChromatography and Lipids. A Practical Guide—Ayr, Scotland: Oily Press,1989, Repr. 1992, IX, 307 S. (Oily Press Lipid Library; 1); “Progress inLipid Research, Oxford: Pergamon Press, 1 (1952)-16 (1977) under thetitle: Progress in the Chemistry of Fats and Other Lipids CODEN.

Thus, fatty acids or triacylglycerol (=TAG, abbreviations indicated inbrackets) may be analyzed, for example, by means of fatty acid methylesters (=FAME), gas liquid chromatography-mass spectrometry (=GC-MS) orthin-layer chromatography (TLC).

Unequivocal proof of the presence of fatty acid products may be obtainedby means of analyzing recombinant organisms following standardanalytical procedures: GC, GC-MS or TLC, as variously described byChristie and references therein (1997, in: Advances on LipidMethodology, fourth ed.: Christie, Oily Press, Dundee, 119-169; 1998,Gaschromatographie-Massenspektrometrie-Verfahren [Gaschromatography-mass spectrometry methods], Lipide 33:343-353).

The plant material to be analyzed may for this purpose be disruptedeither by sonification, glass milling, liquid nitrogen and grinding orvia other applicable processes. After the material has been disrupted,it is then centrifuged. The sediment is then resuspended in distilledwater, heated at 100° C. for 10 min, cooled on ice and centrifugedagain, followed by extraction in 0.5 M sulfuric acid in methanolcontaining 2% dimethoxypropane for 1 h at 90° C., leading to hydrolyzedoil and lipid compounds which result in transmethylated lipids. Thesefatty acid methyl esters may then be extracted in petroleum ether andfinally be subjected to GC analysis using a capillary column (Chrompack,WCOT Fused Silica, CP-Wax-52 CB, 25 μm, 0.32 mm), with a temperaturegradient of between 170° C. and 240° C. for 20 min and at 240° C. for 5min. The identity of the resulting fatty acid methyl esters can bedefined using standards available from commercial sources (i.e. Sigma).

In the case of fatty acids for which no standards are available, theidentity may be shown via derivatization and subsequent GC-MS analysis.For example, the localization of triple-bond fatty acids is shown viaGC-MS after derivatization with 4,4-dimethoxyoxazoline derivatives(Christie, 1998, see above).

B) Fatty Acid Analysis in Plants

Total fatty acids were extracted from plant seeds and analyzed by meansof gas chromatography.

The seeds were taken up with 1% sodium methoxide in methanol andincubated at RT (approx. 22° C.) for 20 min. This was followed bywashing with NaCl solution and taking up the FAMEs in 0.3 ml of heptane.

The samples were fractionated on a ZEBRON-ZB Wax capillary column (30 m,0.32 mm, 0.25 μm; Phenomenex) in a Hewlett Packard 6850 gaschromatograph with flame ionization detector. The oven temperature wasprogrammed from 70° C. (hold for 1 min) to 200° C. at a rate of 20°C./min, then to 250° C. (hold for 5 min) at a rate of 5° C./min andfinally to 260° C. at a rate of 5° C./min. The carrier gas used wasnitrogen (4.5 ml/min at 70° C.). The fatty acids were identified bycomparison with retention times of FAME standards (SIGMA).

Example 4 Functional Characterization of CeLPLATs in Yeast

a) Heterologous Expression in Saccharomyces cerevisiae

To characterize the function of the C. elegans CeLPLATs (SEQ ID NO:38-44), the open reading frames of the particular cDNAs were cloneddownstream of the galactose-inducible GAL1 promoter of pYes2.1Topo,using the pYes2.1TOPO TA Expression Kit (Invitrogen), resulting inpYes2-T06E8.1 and pYes2-F59F4.4.

Since expression of the CeLPLATs should result in an efficient exchangeof the acyl substrates, the double construct pESCLeu-PpD6-Pse1 whichincludes the open reading frames of a Δ-6-desaturase (PpD6) and aΔ-6-elongase (PSE1) from Physcomitrella patens (see DE 102 19 203) wasalso prepared. The nucleic acid sequence of said Δ-6-desaturase (PpD6)and said Δ-6-elongase (Pse1) are indicated in each case in SEQ ID NO: 46and SEQ ID NO: 48. The corresponding amino acid sequences can be foundin SEQ ID NO: 47 and SEQ ID NO: 49.

The Saccharomyces cerevisiae strains C13ABYS86 (protease-deficient) andINVSc1 were transformed simultaneously with the vectors pYes2-T06E8.1and pESCLeu-PpD6-Pse1 and, respectively, pYes2-F59F4.4 andpESCLeu-PpD6-Pse1 by means of a modified PEG/lithium acetate protocol.The control used was a yeast which was transformed with thepESCLeu-PpD6-Pse1 vector and the empty vector pYes2. The transformedyeasts were selected on complete minimal medium (CMdum) agar platescontaining 2% glucose but no uracil or leucine. After selection, 4transformants, two pYes2-T06E8.1/pESCLeu-PpD6-Pse1 and twopYes2-F59F4.4/pESCLeu-PpD6-Pse1 and one pESCLeu-PpD6-Pse1/pYes2 wereselected for further functional expression. The experiments describedwere also carried out in the yeast strain INVSc1.

In order to express the CeLPAATs, precultures of in each case 2 ml ofCMdum liquid medium containing 2% (w/v) raffinose but no uracil orleucine were first inoculated with the selected transformants andincubated at 30° C., 200 rpm, for 2 days. 5 ml of CMdum liquid medium(without uracil and leucine) containing 2% raffinose, 1% (v/v) TergitolNP-40 and 250 μM linoleic acid (18:2^(Δ9,12)) or linolenic acid(18:3^(Δ9, 12, 15)) were then inoculated with the precultures to anOD₆₀₀ of 0.08. Expression was induced at an OD₆₀₀ of 0.2-0.4 by adding2% (w/v) galactose. The cultures were incubated at 20° C. for a further48 h.

Fatty Acid Analysis

The yeast cells from the main cultures were harvested by centrifugation(100×g, 10 min, 20° C.) and washed with 100 mM NaHCO₃, pH 8.0 in orderto remove residual medium and fatty acids. Fatty acid methyl esters(FAMEs) were prepared from the yeast cell sediments by acidicmethanolysis. For this, the cell sediments were incubated with 2 ml of1N methanolic sulfuric acid and 2% (v/v) dimethoxypropane at 80° C. for1 h. Extraction of the FAMES was carried out by extracting twice withpetroleum ether (PE). Nonderivatized fatty acids were removed by washingthe organic phases in each case once with 2 ml of 100 mM NaHCO₃, pH 8.0and 2 ml of distilled water. The PE phases were subsequently dried withNa₂SO₄, evaporated under argon and taken up in 100 μl of PE. The sampleswere separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 μm,Agilent) in a Hewlett Packard 6850 gas chromatograph with flameionization detector. The conditions for the GLC analysis were asfollows: the oven temperature was programmed from 50° C. to 250° C. at arate of 5° C./min and finally at 250° C. (hold) for 10 min.

The signals were identified by comparing the retention times with thoseof corresponding fatty acid standards (Sigma).

Acyl-CoA Analysis

The acyl-CoA analysis was carried out as described in Larson and Graham(2001; Plant Journal 25: 115-125).

Expression Analysis

FIGS. 2 A and B and FIGS. 3 A and B depict the fatty acid profiles oftransgenic C13ABYS86 yeasts fed with 18:2^(Δ9, 12) and 18:3^(Δ9, 12,15),respectively. The substrates fed can be detected in large amounts in alltransgenic yeasts. All four transgenic yeasts display synthesis of18:3^(Δ6,9,12) and 20:3^(Δ8,11,14) and, respectively, 18:4^(Δ6,9,12,15)and 20:4^(Δ8,11,14,17) the products of the Δ-6-desaturase andΔ-6-elongase reactions, meaning that the genes PpD6 and Pse1 were ableto be functionally expressed.

FIG. 3 depicts, as described above, the fatty acid profiles oftransgenic C13ABYS86 S. cerevisiae cells. The fatty acid methyl esterswere synthesized by acidic methanolysis of intact cells which had beentransformed either with the pESCLeu-PpD6-Pse1/pYes2 (A) or with thepYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B) vectors. The yeasts were cultured inminimal medium in the presence of 18:2^(Δ9, 12). The fatty acid methylesters were subsequently analyzed by GLC.

In the control yeasts transformed with the pESCLeu-PpD6-Pse1/pYes2vectors, the proportion of 20:3^(Δ8,11,14) to which 18:3^(Δ6,9,12) iselongated by Pse1 is substantially lower than in the yeasts whichadditionally express LPLAT T06E8.1. In fact, elongation of18:3^(Δ6,9,12) and 18:4^(Δ6,9,12,15) was improved by 100-150% byadditional expression of CeLPLAT (T06E8.1) (FIG. 4). This significantincrease in the LCPUFA content can be explained only as follows: theexogenously fed fatty acids (18:2^(Δ9, 12) and 18:3^(Δ9, 12, 15),respectively) are first incorporated into phospholipids and desaturatedthere by Δ-6-desaturase to give 18:3^(Δ6,9,12) and 18:4^(Δ6,9,12,15).Only after reequilibration with the acyl-CoA pool can 18:3^(Δ6,9,12) and18:4^(Δ6,9,12,15) be elongated by the elongase to give 20:3^(Δ8,11,14)-and 20:4^(Δ8,11,14,17)-CoA, respectively and then incorporated againinto the lipids. LPLAT T06E8.1 is capable of converting theΔ-6-desaturated acyl groups very efficiently back to CoA thioesters.Interestingly, it was also possible to improve the elongation of the fedfatty acids 18:2^(Δ9, 12) and 18:3^(Δ9,12,15). (FIGS. 2A and B and FIGS.5 A and B, respectively).

FIG. 5 indicates the fatty acid profiles of transgenic C13ABYS86 S.cerevisiae cells. Synthesis of the fatty acid methyl esters was carriedout by acidic methanolysis of intact cells which had been transformedeither with the vectors pESCLeu-PpD6-Pse1/pYes2 (A) or with the vectorspYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B). The yeasts were cultured in minimalmedium in the presence of 18:3^(Δ9, 12, 15). The fatty acid methylesters were subsequently analyzed via GLC.

In contrast, expression of a different CeLPLAT (F59F4.4) has noinfluence on elongation (FIG. 4). F59F4.4 evidently does not code for anLPLAT. Thus, not every putative LPLAT nucleic acid sequence isenzymatically active in the reaction found according to the invention.

FIG. 4 indicates the elongation of exogenously applied 18:2^(Δ9, 12) and18:3^(Δ9,12,15), following their endogenous Δ-6-desaturation (data ofFIGS. 2 and 5). The exogenously fed fatty acids are first incorporatedinto phospholipids and desaturated there to give 18:3^(Δ6,9,12) and18:4^(Δ6,9,12,15). Only after reequilibration with the acyl-CoA pool can18:3^(Δ6,9,12) and 18:4^(Δ6,9,12,15) be elongated by the elongase togive 20:3^(Δ8,11,14)- and 20:4^(Δ8,11,14,17)-CoA, respectively, and thenincorporated again into the lipids. LPLAT T06E8.1 is capable ofconverting the Δ-6-desaturated acyl groups efficiently back toCoA-thioesters.

These results show that CeLPLAT (T06E8.1), after coexpression withΔ-6-desaturase and Δ-6-elongase, leads to efficient production ofC20-PUFAs. These results can be explained by the fact that CeLPLAT(T06E8.1) makes possible an efficient exchange of the newly synthesizedfatty acids between lipids and the acyl-CoA pool (see FIG. 6).

FIG. 6 indicates the acyl-CoA composition of transgenic INVSc1 yeaststransformed with the pESCLeu PpD6Pse1/pYes2 (A) orpESCLeu-PpD6-Pse1/pYes2-T06E8.1 (B) vectors. The yeast cells werecultured in minimal medium without uracil and leucine in the presence of250 μM 18:2^(Δ9,12). The acyl-CoA derivatives were analyzed via HPLC.

When using the yeast strain INVSc1 for coexpression of CeLPLAT (T06E8.1)together with PpD6 and Pse1, the following picture emerges: controlyeasts expressing PpD6 and Pse1 comprise, as already shown when usingthe strain C13ABYS86, only small amounts of the elongation product(20:3^(Δ8,11,14), with 18:2 feed, and 20:4^(Δ8,11,14,17) with 18:3 feed;see FIGS. 7 A and 8 A, respectively). Additional expression of CeLPLAT(T06E8.1) results in a marked increase in these elongation products (seeFIGS. 7 B and 8 B). Table 5 indicates that additional expression ofCeLPLAT surprisingly causes an 8-fold increase in the 20:3^(Δ8,11,14)(with 18:2 feed) and, respectively, the 20:4^(Δ8,11,14,17) (with 18:3feed) content. It is also revealed that C16:2^(Δ6,9) is also elongatedmore efficiently to give C18:2^(Δ6,9).

The fatty acid profiles of transgenic INVSc1 S. cerevisiae cells can beseen from FIG. 7. Synthesis of the fatty acid methyl esters was carriedout by acid methanolysis of intact cells which had been transformedeither with the vectors pESCLeu-PpD6-Pse1/pYes2 (A) orpYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B). The yeasts were cultured in minimalmedium in the presence of 18:2^(Δ9,12). The fatty acid methyl esterswere subsequently analyzed via GLC.

The fatty acid profiles of transgenic INVSc1 S. cerevisiae cells can beseen from FIG. 8. Synthesis of the fatty acid methyl esters was carriedout by acid methanolysis of intact cells which had been transformedeither with the vectors pESCLeu-PpD6-Pse1/pYes2 (A) orpYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B). The yeasts were cultured in minimalmedium in the presence of 18:3^(Δ12,15). The fatty acid methyl esterswere subsequently analyzed via GLC.

TABLE 5 Fatty acid composition (in mol %) of transgenic yeaststransformed with the pESCLeu PpD6Pse1/pYes2 (PpD6 Pse1) orpESCLeu-PpD6-Pse1/pYes2- T06E8.1 (PpD6 Pse1 + T06E8) vectors. The yeastcells were cultured in minimal medium without uracil and leucine in thepresence of 250 μM 18:2 ^(Δ9,12) or 18:3 ^(Δ9,12,15). The fatty acidmethyl esters were obtained by acidic methanolysis of whole cells andanalyzed via GLC. Each value indicates the average (n = 4) ± standarddeviation. Feeding with 250 μM 18:2 ^(Δ9,12) Feeding with 250 μM 18:3^(Δ9,12,15) Fatty acids PpΔ6/Pse1 PpΔ6/Pse1 + T06E8 PpΔ6/Pse1PpΔ6/Pse1 + T06E8 16:0 15.31 ± 1.36 15.60 ± 1.36 12.20 ± 0.62 16.25 ±1.85 16:1 ^(Δ9) 23.22 ± 2.16 15.80 ± 3.92 17.61 ± 1.05 14.58 ± 1.93 18:0 5.11 ± 0.63  7.98 ± 1.28  5.94 ± 0.71  7.52 ± 0.89 18:1 ^(Δ9) 15.09 ±0.59 16.01 ± 2.53 15.62 ± 0.34 15.14 ± 2.61 18:1 ^(Δl11)  4.64 ± 1.0911.80 ± 1.12  4.56 ± 0.18 13.07 ± 1.66 18:2 ^(Δ9,12) 28.72 ± 3.25 14.44± 1.61 — — 18:3 ^(Δ6,9,12)  3.77 ± 0.41  4.72 ± 0.72 — — 18:3^(Δ9,12,15) — — 32.86 ± 1.20 14.14 ± 2.52 18:4 ^(Δ6,9,12,15) — —  5.16 ±1.04  3.31 ± 1.15 20:2 ^(Δ11,14)  2.12 ± 0.86  4.95 ± 4.71 — — 20:3^(Δ8,11,14)  1.03 ± 0.14  8.23 ± 1.59 — — 20:3 ^(Δ11,14,17) — —  4.12 ±1.54  6.95 ± 2.52 20:4^(Δ8,11,14,17) — —  1.34 ± 0.28  8.70 ± 1.11

A measure for the efficiency of LCPUFA biosynthesis in transgenic yeastis the quotient of the content of the desired Δ-6-elongation productafter Δ-6-desaturation (20:3^(Δ8,11,14) and 20:4^(Δ8,11,14,17),respectively) to the content of fatty acid fed in (18:2^(Δ9,12) and18:3^(Δ9,12,15), respectively). This quotient is 0.04 in INVSc1 controlyeasts expressing PpD6 and Pse1, and 0.60 in yeasts expressing CeLPLATin addition to PpD6 and Pse1. In other words: the content of desiredΔ-6-elongation product after Δ-6-desaturation with coexpression ofCeLPLAT is 60% of the content of the fatty acid fed in in each case. Incontrol yeasts, this content is only approx. 4%, meaning a 15-foldincrease in the efficiency of LCPUFA biosynthesis in transgenic yeastdue to coexpression of LPLAT.

Interestingly, coexpression of CeLPLAT causes not only an increase inthe elongation products mentioned, 20:3^(Δ8,11,14) and20:4^(Δ8,11,14,17) also an increase in the 20:3^(Δ8,11,14):20:2^(Δ11,14)ratio and the 20:4^(Δ8,11,14,17):20:3^(Δ11,14,17) ratio, respectively.This means that, in the presence of LPLAT, Δ-6-elongase preferably usespolyunsaturated fatty acids (18:3^(Δ6,9,12) and 18:4^(Δ6,9,12,15)) assubstrate, while no distinct substrate specificity is discernible in theabsence of LPLAT (18:2^(Δ9,12) and 18:3^(Δ9,12,15) are also elongated).The reason for this may be protein-protein interactions betweenΔ-6-elongase, Δ-6-desaturase and LPLAT or posttranslationalmodifications (partial proteolysis, for example). This will also explainwhy the above-described rise in Δ-6-elongation products withcoexpression of Δ-6-desaturase, Δ-6-elongase and LPLAT is smaller when aprotease-deficient yeast strain is used.

Acyl-CoA analyses of transgenic INVSc1 yeasts fed with 18:2^(Δ9,12) gavethe following result: no 18:3^(Δ6,9,12)-CoA and 20:3^(Δ8,11,14)-CoA isdetectable in control yeasts expressing PpD6 and Pse1, indicating thatneither the substrate (18:3^(Δ6,9,12)-CoA) nor the product(20:3^(Δ8,11,14-)CoA) of Δ-6-elongase is present in detectable amountsin control yeasts. This suggests that the transfer of 18:3^(Δ6,9,12)from membrane lipids into the acyl-CoA pool does not take place or doesnot take place correctly, meaning that there is hardly any substrateavailable for the Δ-6-elongase present, and this in turn explains thelow elongation product content in control yeasts. INVSc1 yeasts whichexpress CeLPLAT in addition to PpD6 and Pse1 and which had been fed with18:2^(Δ9,12) have substantial amounts of 20:3^(Δ8,11,14)-CoA but not of18:3^(Δ6,9,12)-CoA. This indicates that LPLAT transfers 18:3^(Δ6,9,12)from the membrane lipids to the acyl-CoA pool very efficiently.18:3^(Δ6,9,12)-CoA is then elongated by Δ-6-elongase so that20:3^(Δ8,11,14-)CoA but not any 18:3^(Δ6,9,12)-CoA is detectable.

B) Functional Characterization of the CeLPLATs in Transgenic Plants

Expression of Functional ceLPLAT in Transgenic Plants

DE 102 19 203 describes transgenic plants whose seed oil comprises smallamounts of ARA and EPA, due to seed-specific expression of functionalgenes coding for Δ-6-desaturase, Δ-6-elongase and Δ-5-desaturase. Thevector exploited for transformation of these plants can be found in SEQID NO: 56. In order to increase the content of these LCPUFAs, the geneCeLPLAT (T06E8.1) was additionally expressed in seeds in the transgenicplants mentioned.

For this purpose, the coding region of CeLPLAT was amplified via PCR.

Table 6 indicates the primers used for cloning another ceLPLAT cloneinto binary vectors.

TABLE 6  Nucleotide sequences of the PCR primers for cloning CeLPLAT(T06E8.1) into the binary vector pSUN3 Primer Nucleotide sequenceARe503f* (SEQ ID NO: 91) 5′ TTAAGCGCGGCCGCATGGAGAACTTCTGGTCG 3′ARe504r* (SEQ ID NO: 92) 5′ ACCTCGGCGGCCGCCCTTTTACTCAGATTTC 3′ *f:forward, r: reverse

The PCR product was cloned into a pENTRY vector between USP promoter andOCS terminator. The expression cassette was then cloned into the binarypSUN300 vectors. The vector obtained was referred to as pSUN3CeLPLAT(FIG. 1). In addition, the CeLPLAT coding regions were amplified andcloned between LegB4 promoter and OCS terminator. This vector wasreferred to as pGPTVCeLPLAT (FIG. 9A).

In addition, the CeLPLAT coding region was amplified via PCR and clonedbetween LegB4 promoter and OCS terminator. The PCR primers used for thiswere selected so as for an efficient Kosak sequence to be introducedinto the PCR product. Moreover, the CeLPLAT DNA sequence was modified soas to adapt it to the codon usage of higher plants.

The following primers were used for the PCR:

Forward primer (SEQ ID NO: 93):5′-ACATAATGGAGAACTTCTGGTCTATTGTTGTGTTTTTTCTA-3′Reverse primer (SEQ ID NO: 94):5′-CTAGCTAGCTTACTCAGATTTCTTCCCGTCTTTTGTTTCTC-3′

The PCR product was cloned into the cloning vector pCR Script and clonedvia the restriction enzymes XmaI and SacI into the vector pGPTVLegB4-700. The resulting plasmid was referred to as pGPTVLegB4-700+T06E8.1 (FIG. 9A).

The same PCR product was in addition cloned into a multi-gene expressionvector which already comprised the genes for a Phaeodactylum tricornutumdelta-6-desaturase (SEQ ID NO: 69, amino acid sequence SEQ ID NO: 70)and a P. patens delta-6-elongase. The resulting plasmid was referred toas pGPTV USP/OCS-1,2,3 PSE1(Pp)+D6-Des(Pt)+2AT (T06E8-1) (FIG. 9B). Thesequences of the vector and of the genes can be found in SEQ ID NO: 71,SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74. The Phaeodactylumtricornutum Δ-6-desaturase extends from nucleotide 4554 to 5987 in SEQID NO: 71. The Physcomitrella patens Δ-6-elongase extends fromnucleotide 1026 to 1898 and that of Caenorhabditis elegans LPLAT extendsfrom nucleotide 2805 to 3653 in SEQ ID NO: 71.

Tobacco plants were cotransformed with the pSUN3CeLPLAT vector and thevector described in DE 102 19 203 and SEQ ID NO: 56, which comprisesgenes coding for Δ-6-desaturase, Δ-6-elongase and Δ-5-desaturase, withtransgenic plants being selected using kanamycin.

Tobacco plants were moreover transformed with the pGPTV USP/OCS-1,2,3PSE1(Pp)+D6-Des(Pt)+2AT (T06E8-1) vector [see SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73 and SEQ ID NO: 74].

Linseed was transformed with the pSUN3CeLPLAT vector. The resultingtransgenic plants were crossed with those transgenic linseed plantswhich already comprised small amounts of ARA and EPA, owing tofunctional gene expression of Δ-6-desaturase, Δ-6-elongase andΔ-5-desaturase.

Linseed was furthermore transformed with the pGPTV LegB4-700+T06E8.1vector. The resulting transgenic plants were crossed with thosetransgenic linseed plants which already comprised small amounts of ARAand EPA, owing to functional expression of Δ-6-desaturase, Δ-6-elongaseand Δ-5-desaturase.

The seeds of transgenic tobacco and linseed plants were, as describedhereinbefore [example 3b)], studied for increased LCPUFA contents.

The function of acyl-CoA:lysophospholipid acyltransferase (LPLAT) can bededuced from the studies presented herein as depicted in FIGS. 10 A and10 B. The biosynthetic pathway of LCPUFAS is thus as follows.

Desaturases catalyze the introduction of double bonds into lipid-coupledfatty acids (sn2-acyl-phosphatidylcholine), while the elongasesexclusively catalyze the elongation of coenzyme A-esterified fatty acids(acyl-CoAs). According to this mechanism, the alternating action ofdesaturases and elongases requires continuous exchange of acylsubstrates between phospholipids and acyl-CoA pool and thus theexistence of an additional activity which converts the acyl substratesto the substrate form required in each case, i.e. lipids (fordesaturases) or CoA thioesters (for elongases). This exchange betweenacyl-CoA pool and phospholipids is made possible by LCPUFA-specificLPLAT. The biosynthesis of ARA (A) takes place analogously to that ofEPA (B), but with the difference that, in the case of EPA, aΔ-15-desaturation takes place upstream of the Δ-6-desaturation so thatα8:3-PC acts as a substrate for Δ-6-desaturase. The biosynthesis of DHArequires a further exchange between phospholipids and acyl-CoA pool viaLPLAT: 20:5^(Δ5,8,11,14,17) is transferred from the phospholipids poolto the CoA pool and, after Δ-5-elongation, 22:5^(Δ7,10,13,16,19) istransferred from the CoA pool to the phospholipids pool and finallyconverted by Δ-4-desaturase to give DHA. The same applies to theexchange in the biosynthetic pathway using Δ-8-desaturase, Δ-9-elongaseand Δ-5-desaturase.

Example 5 Functional Characterization of the Acyltransferases

To compare the substrate specificity of acyltransferases of higherplants and LCPUFA-producing organisms, microsomal fractions wereisolated from the LCPUFA-producing organism Mortierella alpina and fromsunflower. The GPAT and LPAAT activities were assayed with differentacyl-CoAs as substrate.

A position analysis of the lipids was carried out to verify whether theLCPUFA producer Thraustochytrium does indeed incorporate DHA at the sn-2position of the lipids.

To isolate LCPUFA-specific acyltransferases, cDNA libraries wereestablished starting from mRNA of the LCPUFA-producing organismsThraustochytrium, Physcomitrella, Cryptecodinium cohnii and Fusarium anda Shewanella genomic library was established, and these libraries wereanalyzed in greater detail via DNA sequencing. Acyltransferase cloneswere identified via sequence homologies. As an alternative,acyltransferases were amplified via PCR techniques.

Transgenic E. coli cells, yeasts, insect cells and plant cells with anelevated expression of at least one LCPUFA-specific acyltransferase havean elevated LCPUFA content in their lipids.

Example 6 Isolation of Microsomal Fractions from Mortierella, Sunflowerand Linseed, and Analysis of the Substrate Specificity ofAcyltransferases for Different Acyl-CoAs

To find out whether higher plants, in particular oil seed plants such assunflower, linseed, oilseed rape or soybean, can incorporate LCPUFAsinto their lipids, microsomes were prepared from sunflower and linseed,and different acyltransferase activities were studied for theirsubstrate specificity for LCPUFA-CoAs. Specifically, GPAT, LPAAT andLPCAT activities were studied. These results were compared with thecorresponding acyltransferase activities of the LCPUFA producersMortierella alpina, which, as is known, comprises high levels of theLCPUFA arachidonic acid in its lipids and in the triacylglycerol (C.Ming et al. (1999) Bioresource Technology 67: 101-110).

Preparation of Microsomal Membranes from Cotyledons of Maturing Seeds ofSunflower and Linseed

All the procedures were carried out at 4° C. The cotyledons of maturingsunflower seeds and linseed were harvested approximately 10 days afteranthesis and suspended in 0.1 M sodium phosphate buffer (pH 7.2),comprising 0.33 M sucrose and 0.1% BSA (free from fatty acids). Aftercomminution in a glass homogenizer, the homogenate was centrifuged for30 minutes at 20 000×g. The supernatant was filtered through one layerof Miracloth and centrifuged for 90 minutes in an ultracentrifuge at 100000×g. The pelleted microsomal membranes were washed with 0.1 M sodiumphosphate buffer (pH 7.2) and resuspended in a small volume of buffer,using a glass homogenizer. The microsomal membrane preparations wereeither immediately processed or stored at −80° C.

Preparation of Microsomal Membranes from Mortierella

Mortierella cultures were harvested after 5 days and placed on ice. Allfurther procedures were carried out at 4° C. The mycelium was suspendedin 0.1 M sodium phosphate buffer (pH 7.2), comprising 0.33 M sucrose,0.1% BSA (free from fatty acids), 1000 units of catalase/ml and 1 mMPefabloc. The following steps were carried out as described under“preparations of microsomal membranes from cotyledons of maturing seedsof sunflower and linseed”.

Acyl-CoA substrate specificity of GPAT: conversion of individualacyl-CoA substrates in the acylation of [¹⁴C] glycerol-3-phosphate

The specificity of the GPAT was studied to verify whether the enzyme hasa preference for certain acyl-CoAs, in particular to determine whetherthe GPAT from oil seed plants converts LCPUFA-CoAs. Microsomal membraneswere incubated with 0.5 mM (Mortierella) or 0.2 mM (sunflower andlinseed) of one of the following acyl-CoAs: myristoyl-CoA (14:0-CoA),palmitoyl-CoA (16:0-CoA), palmitoleoyl-CoA (16:1-CoA), stearoyl-CoA(18:0-CoA), oleoyl-CoA (18:1-CoA), linoleoyl-CoA (18:2-CoA),dihomo-gamma-linolenoyl-CoA (20:3-CoA) or arachidonyl-CoA (20:4-CoA) and5 mM [¹⁴C] G3P. Microsomal membranes (equivalent to 50 μg of protein inthe case of sunflower and Mortierella and 150 μg of protein in the caseof linseed) were added to the reaction mixture in order to start thereaction. After incubation for 5 minutes, the lipids were extracted bythe method of Bligh & Dyer, and the radioactivity incorporated incomplex lipids was determined.

FIG. 11 and table 7a and 7b show the GPAT activities of Mortierella,sunflower and linseed for different acyl-CoA substrates.

The GPAT of Mortierella incorporates unsaturated fatty acids moreefficiently than saturated fatty acids. Oleate and linoleate wereconverted with similar incorporation rates (100% and 90%, respectively).The incorporation of polyunsaturated fatty acids (20:3-CoA and 20:4-CoA)was only marginally lower (80% and 75%, respectively).

Oleate and linoleate are also the best substrates for GPAT in microsomalmembranes (100% and 85% activity, respectively). Acyl-CoAs of thesaturated fatty acids stearate and palmitate are only incorporatedapproximately half as efficiently (40% and 64%, respectively). This alsoapplies analogously for 20:3-CoA (55%). Arachidonyl-CoA is a relativelypoor substrate for sunflower GPAT (23%).

The GPAT in microsomal membranes of linseed has the lowest specificactivity of all GPAT enzymes studied. With 6 nmol/min/mg protein, it isonly half as active as sunflower GPAT and 5 times less active than theenzyme from Mortierella. As regards the substrate specificities behavesThe most efficient acyl-CoA substrates of the linseed GPAT are oleateand linoleate (100% and 90%, respectively), as is the case withsunflower. The incorporation rates of the saturated fatty acids stearateand palmitate, at 65% and 90%, are markedly higher than in the case ofsunflower. In contrast, arachidonyl-CoA is a very poor substrate forlinseed GPAT (5%).

Acyl-CoA Substrate Specificity of LPAAT: Conversion of IndividualAcyl-CoA Substrates in the Acylation of Lysophosphatidic Acid

The specificity of the LPAAT was studied in order to verify whether theenzyme has a preference for certain acyl-CoAs, in particular todetermine whether the LPAAT from oil seed plants converts LCPUFA-CoAs.LPAAT activity was determined in a continuous spectraphotometric assayin which 5,5-dithiobis-2-nitrobenzoate (DTNB) was used, and the changein absorption at 409 nm and 25° C. was monitored (F. M. Jackson et al.(1998) Microbiology 144: 2639-2645). The assay comprisedsn-1-oleoyl-lysophosphatidic acid (30 nmol), DTNB (50 nmol) and 20 nmolof one of the following acyl-CoAs: palmitoyl-CoA (16:0-CoA),stearoyl-CoA (18:0-CoA), oleoyl-CoA (18:1-CoA), linoleoyl-CoA(18:2-CoA), dihomo-gamma-linolenyl-CoA (20:3-CoA) or arachidonyl-CoA(20:4-CoA) in 1 ml of 0.1 M phosphate buffer, pH 7.2. The CoA liberatedin the reaction was determined quantitatively with the aid of theinitial increase and the absorption coefficient of 13.6 mM-1×cm⁻¹.Microsomal membranes (equivalent to 10 μg of protein in the case ofMortierella and 40 μg of protein in the case of sunflower and linseed)were added to the reaction mixture in order to start the reaction.

FIG. 11 and table 7a and 7b show the LPAAT activities of Mortierella,sunflower and linseed for different acyl-CoA substrates.

The Mortierella LPAAT incorporates oleoyl-CoA most efficiently (100%).Linoleoyl-CoA is likewise converted very efficiently (90%). While thesaturated fatty acid substrates 16:0-CoA and 18:0-CoA are onlyincorporated at 40% and 36%, respectively, the LCPUFA substrates20:3-CoA and 20:4-CoA are incorporated with a relatively high efficiency(in each case 65%).

In sunflower microsomal membranes, linoleoyl-CoA is the LPAAT substratewhich is most efficiently incorporated into phosphatidic acid (250%relative to oleoyl-CoA). Both saturated and polyunsaturated acyl-CoAwere poor substrates for sunflower LPAAT (relative activities less than20%).

A very similar picture emerges for linseed LPAAT: linoleoyl-CoA is thebest substrate (120% relative to oleoyl-CoA). Saturated fatty acids arepoor LPAAT substrates (25% and 30% for 16:0-CoA and 18:0-CoA).Arachidonyl-CoA is converted least (19% relative activity).

Acyl-CoA Substrate Specificity of LPCAT: Conversion of IndividualAcyl-CoA Substrates in the Acylation of Lysophosphatidylcholine

In higher plants and fungi, fatty acids are desaturated for theproduction of polyunsaturated fatty acids while esterified withphosphatidylcholine (PC) (A. K. Stobart and S. Stymne (1985) Planta 163:119-125; F. M. Jackson et al. (1998) Microbiology 144: 2639-2645). Theinvolvement of PC in the desaturation of fatty acids also in fungirequires the existence of a functional transfer system for fatty acidsinto and from the sn-2 position of PC, analogously to the system whichhas been described for developing oil seeds (Jackson et al., 1998;Stobart et al., 1983). It is assumed that this transfer of the acylgroup from acyl-CoA to the sn-2 position of PC is catalyzed by LPCAT. Inthe present context, the specificity of LPCAT was studied in order toverify whether the enzyme has a preference for certain acyl-CoAs, inparticular in order to determine whether the oil seed LPCAT convertsLCPUFA-CoAs.

LPCAT activity was determined in a continuous spectraphotometric assayin which 5,5-dithiobis-2-nitrobenzoate (DTNB) was used, and the changein absorption at 409 nm and 25° C. was monitored. The assay comprisedsn-1-palmitolysophosphatidylcholine (30 nmol) as acyl acceptor, DTNB (50nmol) and 20 nmol of one of the following acyl-CoAs: myristoyl-CoA(14:0-CoA), palmitoyl-CoA (16:0-CoA), palmitoleoyl-CoA (16:1-CoA),stearoyl-CoA (18:0-CoA), oleoyl-CoA (18:1-CoA), linoleoyl-CoA(18:2-CoA), dihomo-gamma-linolenyl-CoA (20:3-CoA) or arachidonyl-CoA(20:4-CoA) in 1 ml of 0.1 M phosphate buffer, pH 7.2. The reaction wasstarted by addition of microsomal membrane preparation. The amount ofmicrosomal membrane preparation added was 5 μg (Mortierella andsunflower) or 30 μg (linseed). The CoA liberated in the reaction wasdetermined quantitatively with the aid of the initial increase and theabsorption coefficient of 13.6 mM-1×cm-1 at 409 nm.

FIG. 12 and table 7a and 7b show the LPCAT activities of Mortierella,sunflower and linseed for different acyl-CoA substrates.

The results demonstrate that LPCAT is considerably more active inmicrosomal membranes of sunflower and Mortierella than in the case oflinseed (see tables 10a and 10b). Besides 18:1 (100%), Mortierella LPCATalso converts 18:2 (40%), 20:3 (85%) and 20:4 (90%) with highefficiency. Saturated fatty acids are virtually not converted (relativeactivity less than 25%).

Sunflower LPCAT converts oleoyl-CoA and linoleoyl-CoA with similarefficiency (100% and 120% relative activities, respectively).Palmitoyl-CoA and stearoyl-CoA are poor substrates (relative activityless than 20%). 20:3-CoA and 20:4-CoA are virtually not converted(relative activities less than 5%).

The behavior of linseed LPCAT is similar: while oleoyl-CoA andlinoleoyl-CoA are converted equally efficiently, no LPCAT activity wasdetected for 20:3-CoA and 20:4-CoA.

Discussion of the Data for the Acyl-CoA Specificity of GPAT, LPAAT andLPCAT

The substrate specificity of G3P-acylating enzymes was studiedintensively in order to understand the mechanism of the distribution offatty acids in phospholipids and triacylglycerol. Mammalian microsomalGPAT utilizes saturated and unsaturated acyl-CoAs (Yamada & Okuyama,1978; Haldar et al., 1979; Tamai & Lands, 1974). The same wasdemonstrated for plant microsomal GPATs (Frentzen, 1993; Bafor et al.1990). Jackson et al. (1998) furthermore demonstrated that neither GPATnor LPAAT from the fungus Mucor circinelloides has a pronouncedsubstrate specificity for acyl-CoAs. In the case of Mucor, bothsaturated and unsaturated fatty acids are acylated at both positions. Apurified GPAT from the membrane fraction of Mortierella ramanniana, incontrast, showed a clear preference for oleoyl-CoA in contrast topalmitoyl-CoA (Mishra & Kamisaka, 2001).

In order to study whether GPAT in microsomal membranes from Mortierella,sunflower and linseed has a pronounced specificity for certain acyl-CoAspecies, individual acyl-CoAs were added to the microsomes. TheMortierella GPAT has a similarity with other plant, animal and fungalGPATs in as far as it has a broad specificity for acyl-CoAs, i.e.saturated and unsaturated fatty acids are acylated at the sn-1 positionof G3P. The GPATs from sunflower and linseed microsomal membranes alsoutilize saturated and unsaturated acyl donors in a manner similar towhat has been demonstrated for safflower and turnip rape (Bafor et al.,1990), albeit with a preference for unsaturated fatty acids. In general,the Mortierella GPAT is less discriminating than the sunflower andlinseed enzyme. However, it is noticeable that sunflower and linseedGPATs virtually fails to convert arachidonyl-CoA, whereas theMortierella enzyme acylates arachidonyl-CoA in a highly efficientmanner.

In the second acylation step, Mortierella, sunflower and linseed LPAATis active with sn-1-oleoyl lysophosphatidic acid as acyl acceptor.Similarly to GPAT, Mortierella LPAAT also has a broad specificity foracyl-CoAs. These data resemble those from guinea pig and rat livermicrosomes, where, with the exception of stearoyl-CoA, LPAAT esterifiesall acyl-CoAs with 16 and 18 carbon atoms, independently of the degreeof saturation (Hill and Lands, 1968). In the present work, the sunflowerand linseed LPAATs showed a pronounced specificity for linoleate andoleate. Saturated fatty acids, in contrast, were scarcely converted.These data agree with the observation that, in most oil seed crops,LPAATs show a higher specificity for unsaturated fatty acids (Griffithset al., 1985; Ichihara et al., 1987). In sunflower and linseed,arachidonyl-CoA is a poor substrate, even for LPAAT. In comparison withGPAT, the LPAAT activity of sunflower and linseed is somewhat higher,however.

The specificity of LPCAT in microsomal preparations of Mortierella andsunflower was likewise studied. In Mortierella, LPCAT showed a broadspectrum of substrate specificity. The activity of the enzyme withdifferent acyl-CoAs decreased in the order18:1-CoA>20:4-CoA>20:3-CoA>16:1-CoA>18:2-CoA. Sunflower and linseedLPCAT showed virtually no activity with 20:3 and 20:4-CoA. LPCAT inbovine brain microsomes also showed a weak activity with saturatedacyl-CoAs and a more pronounced activity with linoleoyl- and oleoyl-CoA(Deka et al., 1986). LPCAT from bovine heart muscle microsomes accept awide range of substrates, although the activity is particularly highwith arachidonyl-, linoleoyl- and oleoyl-CoA substrates (Sanjawara etal., 1988). In plants, the acyl specificity and selectivity of LPCAT wasstudied in microsomes of safflower (Stymne et al., 1983; Griffith etal., 1985) and linseed (Stymne & Stobart, 1985a). Oleate and linoleatewere acylated with approximately the same conversion rate at the sn-2position of PC. The activity with alpha-linoleate was only approximatelyhalf as much. Palmitate and stearate were considerably poorer LPCATsubstrates when they were offered as individual acyl-CoAs. If a mixtureof saturated and unsaturated acyl-CoAs was offered, palmitate andstearate were completely excluded by the PC. LPCAT in microsomalmembranes of Mucor circinelloides too utilizes oleoyl- and linoleoyl-CoAmuch more efficiently than saturated fatty acids. There is thus a greatdegree of agreement in the specificity of plant, animal and fungalLPCATs. The fact that LPCAT from Mortierella microsomal membranes onlyshows poor activity with stearoyl-CoA and good activity with oleoyl- andlinoleoyl-CoA might suggest that phosphatidylcholine acts as substratefor desaturases. It was demonstrated that oleate at the sn-1 and thesn-2 position of PC acts as substrate for Δ-12-desaturase in oil seedplants (Stymne & Stobart, 1986; Griffiths et al., 1988). Similar resultswere reported for Mucor circinelloides (Jackson et al., 1998).Δ-6-Desaturase also utilizes linoleate at the sn-2 position of PC inmicrosomal membrane preparations of Mucor (Jackson et al., 1998). TheΔ-6-desaturase from borage, too, utilizes exclusively linoleate at thesn-2 position of the phospholipid (Stymne & Stobart, 1986; Griffiths etal., 1988).

The results described in example 6 demonstrate that acyltransferasesfrom sunflower and linseed are not capable of efficiently incorporatingLCPUFAs such as dihomo-γ-linolenate and arachidonate into the membraneand storage lipids. While LCPUFAs can be produced in oil seed plantssuch as sunflower, linseed or soybean, by functionally expressing thebiosynthetic genes in question, it can be assumed that the resultingLCPUFAs are not efficiently incorporated into triacylglycerol as theresult of lacking acyltransferase activities, which leads to a pooryield. Thus, acyltransferases with a high specificity for LCPUFA-CoAsmust be transformed into oil seed plants in addition to LCPUFAbiosynthetic genes (for example desaturases and elongases or polyketidesynthases). Suitable for this purpose are acyltransferases fromLCPUFA-producing organisms such as Mortierella, Phaeodactylum,Crypthecodinium, Physcomitrella, Euglena and Thraustochytrium.

Table 7a and 7b indicate the activity and acyl specificity of linseed,sunflower and Mortierella alpina acyltransferases.

TABLE 7a Activity and acyl specificity of linseed and sunfloweracyltransferases Linseed Sunflower Enzyme activity GPAT LPAAT LPCAT GPATLPAAT LPCAT Rate (nmol/min/mg protein) of 6 25 9 13 28 360 theincorporation of oleic acid Percentage incorporation in comparison withthe incorporation of oleic acid Myristoyl-CoA 100 30 0 57 16 1 SSAPalmitoyl-CoA 90 25 5 64 15 13 Palmitololeoyl-CoA 140 180 140 90Stearoyl-CoA 65 30 15 40 14 18 Oleoyl-CoA 100 100 100 100 100 100Linoleoyl-CoA 90 120 100 85 250 120 20:3-CoA 0 55 3 Arachidonoyl-CoA 519 0 23 18 4

TABLE 7b Activity and acyl specificity of Mortierella alpinaacyltransferases Mortierella alpina Enzyme activity GPAT LPAAT LPCATRate (nmol/min/mg protein) of 30 51 350 the incorporation of oleic acidPercentage incorporation in comparison with the incorporation of oleicacid Myristoyl-CoA 55 0 Palmitoyl-CoA 66 40 25 Palmitololeoyl-CoA 70 60Stearoyl-CoA 50 36 10 Oleoyl-CoA 100 100 100 Linoleoyl-CoA 90 90 4020:3-CoA 80 65 85 Arachidonoyl-CoA 75 65 90

Example 7 Position Analysis of the Lipids from Thraustochytrium

It was demonstrated in example 6 that LCPUFA producers such asMortierella have membrane-bound acyltransferase activities whichincorporate LCPUFA-CoAs into membrane and storage lipids. Positionanalyses of the lipids from LCPUFA producers allow conclusions to bedrawn regarding the in-vivo activities of the individualacyltransferases. This is why the question of which fatty acids areesterified at the individual positions of the lipids of the DHA producerThraustochytrium was studied herein below.

a) Cultivation of Thraustochytrium spec.(TS) ATCC 26185

Cultivation of the fungus TS was performed in TS liquid culture and bystreaking onto TS plates. Every three weeks, the fungi were transferredto fresh plates, stored for two days at 28° C. and thereafter stored atRT (approx. 23° C.). The liquid culture was incubated with shaking at30° C. and harvested after 6 days. Shaking the culture with exposure tolight increases the lipid yield (data not shown).

I) TS medium: (Bajpai et al. (1991) JAOCS 68: 507-514)a) 10× solution A (g/I):

250 g/l NaCl  50 g/l MgSO₄•7H₂O  10 g/l KCl  20 g/l Na glutamate  2 g/l(NH4)₂SO₄  20 g/l glucoseAutoclave solution.b) 10× solution B (g/I)

200 g/l glucose  20 g/l yeast extractSolution B was filter-sterilized.

c) 10x solution C (g/l) 2 g/l CaCO3To dissolve the CaCO₃, the solution was acidified with HCl andthereafter autoclaved.d) 10× solution D (g/I)

1 g/l KH₂PO₄ 1 g/l NaHCO₃The solution was autoclaved.Supplements: thiamine and vitamin B₁₂

In each case 100 ml of the 10× solutions a) to d) and 10 μg/l thiamineand 1 μg/l vitamin B₁₂ were added to 600 ml of autoclaved distilledwater.

b) Lipid Analysis of Thraustochytrium (Bligh & Dyer (1959) Canadian J.Biochem. 37: 911-917)

To extract the total lipids from TS in liquid culture, the former weresedimented by centrifugation for 10 minutes at 3000 g. Resuspension ofthe cells in 10 ml of 0.45% NaCl was followed by boiling for 10 minutesin a water bath. After a further centrifugation step (as above) of thesuspension, which had been transferred into 40 ml ground-glass tubes,the sediment was taken up in trichloromethane/methanol 1:2 (v/v). Here,the volume of the solvent mixture depended on the volume of thesediment. In general, 10 ml of the mixture were required for extractinga 100 ml culture. The first extraction took place for at least 6 hours,but mostly overnight at 8° C. on a shaker. Thereafter, what remained ofthe cells was resedimented and the supernatant was stored at 8° C. Thesecond extraction was performed analogously to the first extraction,however using trichloromethane/methanol 2:1 (v/v) overnight. After thesecond extraction, what was left of the cells was resedimented, and thesupernatant was combined with that of the first extraction. Then, thecombined extracts were brought to a trichloromethane/methanol/0.45% NaClratio of 2:1:0.7 and shaken. Here, undesired coextracted substances suchas sugars are extracted by shaking and then enter aqueous phase. Then,the extract was centrifuged until phase separation occurred, the organicbottom phase was removed and filtered through cotton wool into around-bottomed flask to remove suspended matter. The lipid extract wasevaporated to dryness on a rotary evaporator, the total lipids weretransferred into trichloromethane/methanol 2:1 (v/v) and into aground-glass tube. Then, the extract was again evaporated to drynessunder nitrogen and finally taken up in trichloromethane/methanol 2:1(v/v) in a defined volume.

c) Lipid Analysis from Thraustochytrium Membranes

Isolated Thraustochytrium membranes were transferred into a ground-glasstube, taken up in 0.45% NaCl and boiled for 5 minutes in a water bath toinactivate lipid-degrading enzymes. After centrifugation (5 minutes,3000×g), the aqueous supernatant was decanted off. The lipids wereextracted for one hour at 4° C. in trichloromethane/methanol (2:1).After addition of ⅓ volume of 0.45% NaCl, the samples were centrifugedto improve phase separation (5 minutes, 3000×g). The lipid-containingbottom phase was removed and concentrated in vacuo. The lipids weretaken up in a suitable volume of trichloromethane.

Directly thereafter, the lipids were applied to silica gel plates(silica gel 60, 20×20 cm, 0.25 mm layer thickness; Merck, Darmstadt) forsubjecting the phospholipids to thin-layer chromatographic separation,together with suitable standards. The mobile phase used wastrichloromethane/methanol/glacial acetic acid/H₂O 91/30/4/4 (v/v/v/v).The development time was 1.5 hours. After the solvent had beenevaporated, the plates were stained with 2″,7″-dichlorofluorescein(Merck, Darmstadt; in 0.3% isopropanol) and visualized under UV light(366 nm).

D) Lipase Digestion of the Thraustochytrium Total Lipids

The enzymatic digestion is performed by means of pancreatic lipase (EC3.1.1.3). The hydrolytic cleavage takes place at the phase boundarybetween fat and water, the enzyme specifically attacking the terminalester bonds in the sn-1 and sn-3 positions in triacylglycerols (TAGs).An intermediary concentration of 1,2- and 2,3-diacyl-sn-glycerols, whichare subsequently digested further to give sn-2 monoacylglycerols, takesplace. Following separation by thin-layer chromatography and recovery ofthe sn-2 monoacylglycerol fraction, the fatty acid composition of theTAGs in the middle position is determined.

50 mg of the total lipid were weighed into a ground-glass tube. Afteraddition of 0.5 ml of Tris buffer, 0.1 ml of CaCl₂ solution and 0.25 mlof bile salt solution (0.05% (w/v) bile salt; Sigma, Deisenhofen), theground tube was sealed. The mixture was mixed for one minute andsubsequently prewarmed for one minute in a water bath at 40° C. in orderto emulsify the sample.

Hydrolysis was effected after addition of pancreatic lipase (EC 3.1.1.3;Sigma, Deisenhofen; 2 mg of lipase per 5 mg of lipid; lipase freshlydissolved in 0.5 ml of Tris buffer) at 38° C. and high shaking frequency(if possible 1200 rpm). After 30 minutes, the reaction was stopped byaddition of 1 ml of HCl (6 N) and 1 ml of ethanol.

The reaction mixture was extracted twice in the centrifuge glass, usingin each case 4 ml of diethyl ether. In doing so, the ether phase, whichwas the top phase, was removed. The aqueous phase which remained wasreextracted with diethyl ether. The formation of emulsions wasadditionally prevented in each extraction step by centrifugation. Thecombined ether phases were washed by shaking with in each case 3 ml ofwater (distilled). The organic phase was transferred into a fresh tubeand dried using sodium sulfate. After centrifugation for 2 minutes at3000×g, the clear supernatant was removed and the sodium sulfate pelletwas again extracted by shaking with diethyl ether, centrifuged as statedabove, and the organic phases were combined. After concentration of theether extract in vacuo, the extract was immediately thereafter appliedto silica gel plates (silica gel 60, 20×20 cm, 0.25 mm layer thickness;Merck, Darmstadt) in order to subject the partial glycerides toseparation by thin-layer chromatography. The mobile phase used wasdiisopropyl ether/glacial acetic acid 40:1 (v/v). The development timewas 35-45 minutes. After evaporation of the solvent, the plates werestained using 2″,7″-dichlorofluorescein (Merck, Darmstadt; in 0.3%isopropanol) and visualized under UV light. The individual lipidfractions were separated in the following order: monoacylglycerols (sn-2MAGs, immediately above the starting line), diacylglycerols (sn-1,2- andsn-2,3-DAGs), free fatty acids (FFA) and the unreacted TAGs.

The MAG band was scraped off from the silica gel plate. The fatty acidcomposition of the TAGs was determined by means of transmethylation,followed by gas-chromatographic separation of the fatty acid methylesters (FAMEs).

Tris buffer:1M Tris/HCl, bring to pH 8.0 using HClCaCl solution2.2% (w/v) CaCl₂

e) Lipase Digestion of the Thraustochytrium Membrane Lipids (Fischer etal., 1973)

The position analysis of the membrane lipids was carried out byenzymatic hydrolysis of the sn-2 ester bond with phospholipase A₂ (EC3.1.1.4).

The isolated membrane lipids were concentrated in vacuo, treated with0.5 ml of hydrolysis buffer and dispersed for 5 minutes using asonicator. Hydrolysis was effected at RT after addition of 50 U ofphospholipase A₂. The reaction was stopped by addition of 4 ml oftrichloromethane/methanol 2:1 (v/v) and 0.45% NaCl. The organic, bottomphase was transferred into a fresh vessel, evaporated on a rotaryevaporator and taken up in 200 μl of trichloromethane/methanol 2:1(v/v).

Directly thereafter, the mixture was applied to silica gel plates(silica gel 60, 20×20 cm, 0.25 mm layer thickness; Merck, Darmstadt) inorder to subject the phospholipids to thin-layer chromatographicseparation. The mobile phase used was trichloromethane/methanol/glacialacetic acid/H₂O 91/30/4/4 (v/v/v/v). The development time was 1.5 hours.After evaporation of the solvent, the plates were stained using2″,7″-dichlorofluorescein (Merck, Darmstadt; in 0.3% isopropanol) andvisualized under UV light. Bands of interest were scraped off from thesilica gel plate, transmethylated and thereafter analyzed in a gaschromatograph.

Hydrolysis Buffer

0.1 M boric acid, pH 8.0 3 mM CaCl₂ 1.4 mM sodium deoxycholatef) Transmethylation of Fatty Acids with Sodium Methylate (Method ofLühs)

After the solvent had been evaporated, or after material had beenscraped from the thin-layer plate (for example in the case of sn-2analysis of the total lipids), lipid samples were treated with 2 ml ofsodium methylate solution for transesterification purposes. The mixturewas shaken thoroughly and, in order to subject the fatty acids totransmethylation, incubated for approximately 30 minutes at roomtemperature. Thereafter, 1.5 ml of isooctane were added and the sampleswere carefully shaken twice. The mixture was stored for 30 minutes at 4°C., during which time the fatty acid methyl esters (FAMEs) enter theisooctane phase. After clear phase separation had occurred, the topphase, which was the isooctane phase, was pipetted into a GC tube andthe sample was analyzed in a gas chromatograph.

Sodium Methylate Solution

5 g of sodium methylate were dissolved in 800 ml of methanol (99%) at50° C., using a magnetic stirrer, and, after cooling, made up to 1000 mlwith isooctane.g) Methylation of Free Fatty Acids with Methanolic Sulfuric Acid

In a Pyrex tube with screw top, 1 ml of 1 N methanolic sulfuric acid wasadded to the concentrated lipid extract. The mixture was incubated forone hour at 80° C. After the mixture had been cooled briefly, it wastreated with 1 ml of 0.9% NaCl and mixed. Thereafter, an equal volume ofhexane was added, and the mixture was mixed thoroughly and incubated at4° C. for 30 minutes until phase separation took place. The hexanephase, which was the top phase, was transferred into a GC tube andanalyzed in a gas chromatograph.

Methanolic Sulfuric Acid

2 ml of dimethoxypropanes and 0.5 M H₂SO₄ were added to 100 ml of(anhydrous) methanol.

H) Gas-Chromatographic Analysis

The following parameters of the gas-chromatographic system weremaintained for the GC analyses:

Equipment type HP 6890 GC Injector HP GC injector Detector flameionization detector (FID), temp. 250° C. Column J&W DW23 50%cyanopropyl/methylsiloxanes, 30 m, 0.5 mm diameter Oven temperature 220°C. Carrier gas hydrogen Autosampler HP 7673, injection volume 1 μl ofsample

I) the Lipid Analysis of the Thraustochytrium Lipids Gave the FollowingResults

Fatty acid composition Lipid fraction 16:0 22:3 ω-3 22:4 ω-3 22:6 ω-3Total TAG 24% 12% 31% 23% TAG sn-2 21% 26% 43% Total membrane lipids 16%13% 23% Membrane lipids sn-2 34% 18% 36%

The results show that Thraustochytrium has a high DHA content in itslipids. With besides palmitate, DHA is the main component of thetriacylglyerols and dominating fatty acid of the membrane lipids. It isnoticeable that DHA is markedly concentrated at the sn-2 position ofboth the triacylglycerol and the membrane lipids: 36-43% of the fattyacids at the sn-2 position is DHA. As a result of this data, it can beassumed that Thraustochytrium has an active LPAAT with a highspecificity for DHA-CoA.

Example 8 Isolation of Total RNA and poly(A)⁺ RNA

Total RNA was isolated from plants such as linseed and oilseed rape etc.by a method described by Logemann et al. (Anal. Biochem. (1987) 163:21). The total RNA can be obtained from the moss Physcomitrella patensfrom protonemal tissue using the GTC method (Reski et al. (1994) Mol.Gen. Genet. 244: 351-359).

A) RNA Isolation from Thraustochytrium, Cryptecodinium and Shewanella:

Frozen algal samples (−70° C.) were comminuted in an ice-cold mortarunder liquid nitrogen to give a fine powder. 2 volumes of homogenizationmedium (12.024 g sorbitol, 40.0 ml 1 M Tris-RC1, pH 9 (0.2 M); 12.0 ml 5M NaCl (0.3 M), 8.0 ml 250 mM EDTA, 761.0 mg EGTA, 40.0 ml 10% SDS weremade up to 200 ml with H₂O and the pH was brought to 8.5) and 4 volumesof phenol comprising 0.2% of mercaptoethanol were added to the frozencell powder at 40-50° C., with thorough mixing. Thereafter, 2 volumes ofchloroform were added and the mixture was stirred vigorously for 15minutes. The mixture was centrifuged for 10 minutes at 10 000 g and theaqueous phase was extracted with phenol/chloroform (2 vol/2 vol) andfinally with chloroform.

The resulting volume of the aqueous phase was treated with 1/20 vol of 4M sodium acetate (pR 6) and 1 vol of isopropanol (ice-cold), and thenucleic acids were precipitated ON(=Overnight) at −20° C. The mixturewas centrifuged for 30 minutes at 10 000 g and the supernatant waspipetted off. This was followed by a wash step with 70% EtOH and anothercentrifugation. The sediment was in Tris borate buffer (80 mM Trisborate buffer, 10 mM EDTA, pH 7.0). Then, the supernatant was mixed with⅓ vol of 8 M LiCl, mixed and incubated for 30 minutes at 4° C. Afterrecentrifugation, the sediment was washed with 70% ethanol andcentrifuged, and the sediment was subsequently dissolved in RNAse-freewater.

Poly(A)+ RNA was isolated using Dyna Beads (Dynal, Oslo, Finland)following the instructions in the manufacturer's protocol.

After the RNA or poly(A}+RNA concentration had been determined, the RNAwas precipitated by addition of 1/10 volume of 3 M sodium acetate, pH4.6, and 2 volumes of ethanol and stored at −70° C.

For the analysis, in each case 20 μg of RNA were separated in aformaldehyde-comprising, 1.5% strength agarose gel and transferred ontonylon membranes (Hybond, Amersham). Specific transcripts were detectedas described by Amasino (Amasino (1986) Anal. Biochem. 152: 304).

Example 9 Construction of cDNA Libraries

To construct the cDNA libraries from Physcomitrella, Thraustochytriumand Fusarium, the first-strand synthesis was carried out using reversetranscriptase from murine leukemia virus (Roche, Mannheim, Germany) andoligo-d(T) primers, while the second-strand synthesis was achieved byincubation with DNA polymerase I, Klenow enzyme and RNAse H cleavage at12° C. (2 hours), 16° C. (1 hour) and 22° C. (1 hour): the reaction wasstopped by incubation at 65° C. (10 minutes) and subsequentlytransferred onto ice. Double-stranded DNA molecules were madeblunt-ended using T4 DNA polymerase (Roche, Mannheim) at 37° C. (30minutes). The nucleotides were removed by means of phenol/chloroformextraction and Sephadex G50 centrifugation columns. EcoRI/XhoI adapters(Pharmacia, Freiburg, Germany) were ligated onto the cDNA ends by meansof T4 DNA ligase (Roche, 12° C., overnight), cut again with XhoI andphosphorylated by incubation with polynucleotide kinase (Roche, 37° C.,30 min). This mixture was subjected to separation on a low-meltingagarose gel. DNA molecules of above 300 base pairs were eluted from thegel, extracted with phenol, concentrated on Elutip D columns (Schleicherand Schüll, Dassel, Germany) and ligated with vector arms and packagedin lambda-ZAPII phages or lambda-ZAP Express phages using the GigapackGold kit (Stratagene, Amsterdam, the Netherlands), using themanufacturer's material and following their instructions.

Example 10 DNA Sequencing and Computer Analysis

cDNA libraries as described in example 9 were used for DNA sequencing bystandard methods, in particular by means of the chain termination methodusing the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reactionkit (Perkin-Elmer, Weiterstadt, Germany). Random individual clones weresequenced after plasmid preparation from cDNA libraries via in-vivo massexcision and retransformation of DH10B on agar plates (details onmaterials and protocol from Stratagene, Amsterdam, the Netherlands).Plasmid DNA was prepared from E. coli overnight cultures which had beengrown in Luria broth with ampicillin (see Sambrook et al. (1989) (ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6)) on a Qiagen DNApreparation robot (Qiagen, Hilden) following the manufacturer'sprotocol. Sequencing primers with the following nucleotide sequenceswere used:

(SEQ ID NO: 95) 5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO: 96)5′-CTAAAGGGAACAAAAGCTG-3′ (SEQ ID NO: 97) 5′-TGTAAAACGACGGCCAGT-3′

The sequences were processed and annotated using the standard softwarepackage EST-MAX, which is commercially available from Bio-Max (Munich,Germany). Using comparative algorithms, and using a search sequence, theBLAST program was used for searching for homologous genes (Altschul etal. (1997) “Gapped BLAST and PSI-BLAST: A new generation of proteindatabase search programs”, Nucleic Acids Res. 25: 3389-3402).

Example 11 Identification of Genes by Means of Hybridization

Gene sequences can be used for identifying homologous or heterologousgenes from cDNA or genomic libraries.

Homologous genes (i.e. full-length cDNA clones which are homologous, orhomologs) can be isolated via nucleic acid hybridization using, forexample, cDNA libraries: depending on the frequency of the gene ofinterest, 100 000 up to 1 000 000 recombinant bacteriophages are platedand transferred onto a nylon membrane. After denaturation with alkali,the DNA is immobilized on the membrane, for example by UV crosslinking.Hybridization is effected under high-stringency conditions. The washsteps and the hybridization are carried out in aqueous solution at aionic strength of 1 M NaCl and a temperature of 68° C. Hybridizationprobes were prepared for example by labeling by means of radioactive(32P) nick transcription (High Prime, Roche, Mannheim, Germany). Thesignals are detected by means of autoradiography.

Partially homologous or heterologous genes which are related, but notidentical, can be identified analogously to the above-described methodusing low-stringency hybridization and wash conditions. The ionicstrength for the aqueous hybridization was usually kept at 1 M NaCl, thetemperature being lowered gradually from 68 to 42° C.

Gene sequences with homologies with only a single domain of, forexample, 10 to 20 amino acids can be isolated using syntheticradiolabeled oligonucleotide probes. Radiolabeled oligonucleotides areprepared by phosphorylating the 5′ end of two complementaryoligonucleotides with T4 polynucleotide kinase. The complementaryoligonucleotides are hybridized with one another and ligated so thatconcatemers are formed. The double-stranded concatemers areradiolabeled, for example by Nick transcription. Hybridization isusually effected under low-stringency conditions, using higholigonucleotide concentrations.

Oligonucleotide hybridization solution:

6×SSC

0.01 M sodium phosphate

1 mM EDTA (pH 8) 0.5% SDS

100 μg/ml denatured salmon sperm DNA0.1% dry skim milk

During the hybridization, the temperature was gradually reduced to 5-10°C. below the calculated oligonucleotide Tm or down to room temperaturemeans RT=23° C. in all experiments, unless otherwise specified),followed by wash steps and autoradiography. Washing was carried out withextremely low stringency, for example 3 wash steps using 4×SSC. Furtherdetails are as described by Sambrook, J., et al. (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, orAusubel, F. M., et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons.

Example 12 Isolation and Cloning of an LPAAT Full-Length Clone fromThraustochytrium

Screening a Thraustochytrium cDNA Library

Analogously to what has been described for example 9, a ThraustochytriumcDNA library was generated. In the next step, the phage library wasconverted into a plasmid library by means of a helper phage, followingthe manufacturer's instruction. The plasmid library was plated on LBmedium, 0.8% agar, 100 mg/l ampicillin and incubated. Grown bacterialcolonies were selected randomly, grown in liquid medium (LB, 100 mg/lampicillin) and sequenced as described in example 10.

The sequences obtained were searched for redundancies, and these wereremoved. This gave rise to an assortment of sequences which describes aunigene set. This sequence set was input into the Pedant database(Biomax AG, Martinsried, Germany). A short sequence section with a lowdegree of similarity to known acyltransferases was found by means ofBLAST analysis, using conserved regions within acyltransferases. Theexisting sequence information was used for generating primers(LPAAT069-5′ and LPAAT069-3′). Using this fragment, the cDNA library wasthen searched for a full-length clone (table 8).

TABLE 8  Sequences of the primers employed Primer Sequence T_(m) (° C.)LPAAT069-5′ 5′-GCT ACA TTG CCA TGG AGC-3′ (SEQ ID NO: 98) 56 LPAAT069-3′5′-GCT ACA AGA GGT CAG GTC G-3′ (SEQ ID NO: 99) 59 ACtrau-5′5′-CTG GAT CCA TGA GCG CGT GGA CGA G-3′ (SEQ ID NO: 100) 69 (52)ACtrau-3′ 5′-TTG GAT CCC AAG AGG TCA GGT CGG A-3′ (SEQ ID NO: 101)66 (54) ACtrau-3′stop 5′-TTG GAT CCC TAC AAG AGG TCA GGT CG-3′(SEQ ID NO: 102) 66 (48) YES-HIS-5′ 5′-CTG AGC TCA TGA GCG CGT GGA G-3′(SEQ ID NO: 103) 69 (56) YES-HIS-3′5′-ATG GAT CCG TGA TGG TGA TGG TGA TGC AAG AGG TC-3′ 72 (40)(SEQ ID NO: 104) The melting point T_(m) (° C.)of the oligonucleotideswas calculated by the method of Suggs et al. (1981): T_(m) (° C) = 4(G + C) + 2 (A + T) T_(m) values in brackets refer to actually bindingnucleotides of primers whose ends have been modified by additionallyintroduced cleavage sites.

In the PCR experiments, the constituents of a PCR standard mix, shownhereinbelow, were pipetted into a PCR reaction vessel on ice, placedinto the thermoblock, and the temperature profile shown hereinbelow wasstarted. The polymerase employed was in almost all cases Taq polymerase(Gibco BRL), with Pfu polymerase (Stratagene) only being used foramplifications for the purposes of functional expression in E. coliJC201. In all experiments, the polymerase was added via what is known asa “hot start”, where the enzyme is added only after the DNA template hasbeen subjected to denaturation for 5 minutes. The annealing temperatures(T_(a)) were chosen to be 3-5° C. below the mean melting point T_(m) ofthe primer pairs.

PCR standard mix (for Taq polymerase)5 μl 10×PCR buffer (100 mM Tri-HCl, pH 8.3; 15 mM MgCl₂, 500 mM KCl)1 μl dNTP mix (10 mM dATP, dGTP, dTTP and dCTP)1 μl primer 1 (30 μM)1 μl primer 2 (30 μM)1 U Taq polymerase50-100 ng plasmid DNA templatemake up to 50 μl with distilled water

Hot-Start Program

1. denaturation 95° C., 5 min2. hot start 25° C., 3 min→addition of the polymerase3. denaturation 94° C. 30 s4. annealing T_(m)-5° C., 30 s5. polymerization 72° C., 1-3 min (approx. 60 s for 1.0 kbp)Steps 3. to 5. were repeated cyclically 25 to 30 times.6. polymerization 72° C., 5 min7. termination 4° C.

a) Cold Labeling of DNA

DNA probes were cold-labeled using the “PCR DIG PROBE SYNTHESIS KIT”(Boehringer Mannheim). To do so, DNA fragments were labeled in a PCRreaction with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP).The detection was subsequently carried out by means of ananti-digoxygenin antibody which is conjugated with alkaline phosphatase,and addition of chemiluminescence or color substrates.

To avoid background signals which can be attributed to vector sequences,the PCR labeling first involved, in a first PCR, the amplification ofthe desired DNA with unlabeled dNTPs, the linear fragment was purifiedvia an agarose gel and used as template for the actual PCR labeling, inwhich, in turn, the primer pair of the first PCR was employed. Thelabeling reaction was carried out as specified in the manufacturer'sinstructions. The chosen primer combinations are compiled in the tablewhich follows.

Primer Sequence LPAAT069-5′ 5′-GCT ACA TTG CCA TGG AGC-3′(SEQ ID NO: 105) LPAAT069-3′ 5′-GCT ACA AGA GGT CAG GTC G-3′(SEQ ID NO: 106)b) Screening a cDNA Library

To isolate a complete clone, a Thraustochytrium cDNA library (inλTriplEx2) was searched with the DIG-labeled probe. The probe wasgenerated using the primers LPAAT069-3″ and LPAAT069-5, derived from theEST clone s_(—)3002038069 known cDNA sequence which might code for aThraustochytrium LPAAT.

5×10⁴ plaques were plated in each case on 10 large NZY plates, followingthe manufacturer's instructions (Stratagene). To transfer the phagesonto nitrocellulose filters (Hybond™-C, Amersham), the filters wereplaced on the plates for 1 minute, and their precise position was markedby 3 stamps with a cannula. The filters, stamped side uppermost, weresubsequently treated first for 5 minutes with denaturation solution,then for 5 minutes with neutralization solution and finally for 15minutes with 2×SSC solution. This was carried out using 3 sheets ofWhatman 3 mM paper which had been impregnated with the solutions. Afterthe filters had dried for 5 minutes, the DNA was immobilized by UVtreatment with 0.12 Joule/cm² (UV-Crosslinker, Hoefer ScientificInstruments). Hybridization and colorimetric detection were carried outusing the “Dig System für Filter Hybridisierung” from Boehringer(Mannheim) in accordance with the manufacturer's instructions. Thehybridization buffers used were standard buffers, the hybridizationbeing carried out in 80 ml of hybridization buffer using 15 μl of theprobe PCR mix. After detection had been effected, the precise positionof the signals and the three reference points of the filters weretransferred to plastic films in order to identify the positive plaqueson the plates, using the former as stencil. The positive plaques werethen excised using a flamed cork borer (diameter 5 mm), transferred into1 ml of SM buffer supplemented with 20 μl of CHCl₃, and the phages wereeluted from the agar plugs overnight at 4° C. Accurate excision of theplaques was almost impossible as the result of their high density andsmall size. This is why, as a rule, one to two rescreens are carriedout. In this case, the phage lysates were studied for approx. 570 bpfragments by means of PCR and the primers LPAAT069-3″ and LPAAt-5. Tothis end, aliquots of the phage lysates were treated with EDTA (finalconcentration 10 mM), and 1 μl of this was employed as template for thePCR. Using positive lysates, in-vivo excisions were carried out asspecified in the “ZAP-cDNA® Gigapack® II Gold Cloning Kit” (Stratagene),but instead of the 10-50 μl as stated, only 2 μl of the infected SOLRcells were plated onto LB-Amp plates and incubated overnight at 37° C.The plasmids from the resulting colonies were analyzed directly by meansof PCR and the primers LPAAT-3′ and LPAAT-5′. To this end, pools weregenerated by rubbing in each case 6 colonies into 20 μl of distilledwater in an Eppendorf tube, using sterile toothpicks, and the tubes weresubjected to 3× freeze-thaw cycles in order to lyze the cells,centrifuged for 5 minutes at 14 000×g, and 2 μl of the supernatant wasemployed as template in the PCR reaction. Positive pools were isolated,and the plasmids were isolated via plasmid minipreps and analyzed viaPCR, restriction analyses and DNA sequencing reactions.

Finally, a Thraustochytrium LPAAT full-length clone was identified; itsDNA sequence is shown in SEQ ID NO: 1. The derived amino acid sequenceis shown in SEQ ID NO: 2.

NZY medium (per liter, NZY plates made with 15 g agar)

5 g NaCl

5 g yeast extract10 g NZ amine (casein hydrolysate)

pH 7.5 (NaOH)

2 g MgSO₄ (filter-sterilized)

Denaturation Solution 0.5 M NaOH 1.5 M NaCl Neutralization Solution 1.0M Tris-HCl, pH 7.5 1.5 M NaCl 20×SSC 3.0 M NaCl

0.3 M sodium citrate, pH 7.0

Standard Buffer 5×SSC

0.1% (w/v) N-laurylsarcosine0.02% (w/v) SDS1% blocking reagent

SM Buffer (Per Liter) 5.8 g NaCl 2 g MgSO₄ 50 ml 1 M Tris-HCl, pH 7.5

5 ml 2% strength gelatin

Example 13 Isolation and Cloning of Full-Length Clones for PUFA-SpecificAcyltransferases from Physcomitrella patens, Mortierella alpina andShewanella hanedai

RNA was isolated, and a cDNA library generated, from Physcomitrellapatens and Mortierella alpina as described in examples 8 and 9.

In the next step, the phage library was converted into a plasmid libraryby means of a helper phage, following the manufacturer's instructions.The plasmid library was plated on LB medium, 0.8% agar, 100 mg/lampicillin and incubated. Grown bacterial colonies were selectedrandomly, grown in liquid medium (LB, 100 mg/l ampicillin) and sequencedas described in example 10.

The sequences obtained were searched for redundancies, and these wereremoved. This gave rise to an assortment of sequences which describes aunigene set. This sequence set was input into the Pedant database(Biomax AG, Martinsried, Germany). Short sequence sections with a lowdegree of similarity to known acyltransferases were found by means ofBLAST analysis, using conserved regions within acyltransferases (table9). The existing sequence information was used for generating primers(table 10). Using these primers, the full-length clone was amplified.

For the Shewanella hanedai acyltransferase, the public database ofShewanella putrefaciens MR1 (TIGR databasehttp://tigrblast.tigr.org/ufmg/) was searched for acyltransferases. Asequence with homology to acyltransferases was found in the database. APCR fragment of this sequence was generated by means of standard primersT7 and T3. The resulting product was illustrated as in example 10a) andb), labeled and employed for searching a genomic Shewanella hanedailibrary.

Shewanella hanedai genomic DNA was isolated by the following protocol:

A 100 ml culture was grown at 30° C. to an optical density of 1.0. 60 mlof this were centrifuged for 3 minutes at 3000×g. The pellet wasresuspended in 6 ml of twice-distilled H₂O and divided between 1.5 mlvessels, centrifuged, and the supernatant was discarded. The pelletswere resuspended and lyzed by vortexing with 200 μl of solution A, 200μL of phenol/chloroform (1:1) and 0.3 g of glass beads. After additionof 200 μl of TE buffer pH 8.0, the mixture was centrifuged for 5minutes. The supernatant was subjected to ethanol precipitation with 1ml of ethanol. After the precipitation, the resulting pellet wasdissolved in 400 μl of TE buffer pH 8.0+30 μg/ml Rnase A. Afterincubation for 5 minutes at 37° C., 18 μl of 3 M sodium acetate solutionpH 4.8 and 1 ml of ethanol were added, and the precipitated DNA waspelleted by centrifugation. The DNA pellet was dissolved in 25 μl oftwice-distilled H₂O. The concentration of the genomic DNA was determinedby its absorption at 260 nm.

Solution A: 2% Trition-X100 1% SDS 0.1 M NaCl 0.01 M Tris-HCl pH 8.00.001 M EDTA

The resulting genomic DNA was incubated with the restriction enzymeSau3A (New England Biolabs) for 1 hour at 25° C. following themanufacturer's instructions. The resulting fragments were then ligatedinto a BamHI-digested pUC18 plasmid, using T4 ligase (Roche). Theresulting library was then searched in the same manner as described inexample 10. A clone comprising a 1.7 kb genomic fragment and having a687 bp coding sequence with similarity to acyltransferases was found.

The Shewanella hanedai sequence has a particularly high degree ofsimilarity to the Chaenorabdidis elegans LPCAT. The similarity of thetwo sequences at the amino acid level is 26%.

TABLE 9 Identified acyltransferase from the abovementioned cDNAlibraries Clone No. Organism Homology with MaLPAAT1.1 M. alpina LPAATMaLPAAT1.2 M. alpina LPAAT ShLPAAT S. hanedai LPAAT T6 Thrausto. LPAATpp004064045r P. patens LPAAT pp020064227r P. patens LPAAT pp015052144rP. patens GPAT/LPAT pp004034225r P. patens GPAT pp004104272r P. patensCa-LPAAT pp020018156r P. patens Ca-LPAAT pp015034341r P. patens LPAATpp015033362r P. patens LCAT Fg003028298 Fusarium LCAT

TABLE 10  Sequences of the primers employed: Clone No. OrganismPrimer sequence in 5′-3′ orientation Length in bp MaLPAAT1.1 M. alpinaatggatgaatccaccacgacca (SEQ ID NO: 123) 1254tcagcccgatgcttgctgc (SEQ ID NO: 124) MaLPAAT1.2 M. alpinaatgaaccctatctacaagggt (SEQ ID NO: 125) 1170tcagcccgatgcttgctgc (SEQ ID NO: 126) ShLPAAT S. hanedaiatgttactgctagcatttgt (SEQ ID NO: 127) 687ttactttgccattaagg (SEQ ID NO: 128) T6 Thrausto.atgagcgcgtggacgagggc (SEQ ID NO: 129) 918ctacaagaggtcaggtcggacgtaca (SEQ ID NO: 130) Pp00406404 P. patensatggctttgatgtatatctg (SEQ ID NO: 131) 714ttacacgatttttcttttag (SEQ ID NO: 132) Pp02006422 P. patensatgctgatattacagcccttc (SEQ ID NO: 133) 657ctaatgaacaggaagaccgt (SEQ ID NO: 134) Pp01505214 P. patensatgatccggattttcagag (SEQ ID NO: 135) 444tcagtccgttttgccgaggt (SEQ ID NO: 136) Pp00403422 P. patensatgccgtcgctgtttcggg (SEQ ID NO: 137) 1305tcaatcagttcgcctgcttc (SEQ ID NO: 138) Pp00410427 P. patensatgctgatattacagcccttc (SEQ ID NO: 139) 1566ctaatgaacaggaagaccgt (SEQ ID NO: 140) Pp02001815 P. patensatgaccagcacggaaaatac (SEQ ID NO: 141) 1560ctagatgttagtttcactc (SEQ ID NO: 142) Pp01503434 P. patensatgattatgatggaggtgctg (SEQ ID NO: 143) 1014tcagtccgttttgccgagg (SEQ ID NO: 144) Pp01503336 P. patensatgtgttcaatttcttgtgg (SEQ ID NO: 145) 1503ttagtggaacataagctgtt (SEQ ID NO: 146) Fg003028298 Fusariumatgggaaagtccactttac (SEQ ID NO: 147) 1893ctatgaagtctcctcatcatcg (SEQ ID NO: 148)

In the PCR experiments, the constituents of a PCR standard mix, shownhereinbelow, were pipetted into a PCR reaction vessel on ice, placedinto the thermoblock, and the temperature profile shown hereinbelow wasstarted. The polymerase employed was in almost all cases Taq polymerase(Gibco BRL), with Pfu polymerase (Stratagene) only being used foramplifications for the purposes of functional expression in E. coliJC201. In all experiments, the polymerase was added via what is known asa “hot start”, where the enzyme is added only after the DNA template hasbeen subjected to denaturation for 5 minutes. The annealing temperatures(T_(a)) were chosen to be 3-5° C. below the mean melting point T_(m) ofthe primer pairs.

PCR Standard Mix (for Taq Polymerase)

5 μl 10×PCR buffer (100 mM Tri-HCl, pH 8.3; 15 mM MgCl₂, 500 mM KCl)1 μl dNTP mix (10 mM dATP, dGTP, dTTP and dCTP)1 μl primer 1 (30 μM)1 μl primer 2 (30 μM)1 U Taq polymerase50-100 ng plasmid DNA templatemake up to 50 μl with distilled water

Hot-Start Program

1. denaturation 95° C., 5 min2. hot start 25° C., 3 min→addition of the polymerase3. denaturation 94° C. 30 s4. annealing T_(m)-5° C., 30 s5. polymerization 72° C., 1-3 min (approx. 60 s for 1.0 kbp)Steps 3. to 5. were repeated cyclically 25 to 30 times.6. polymerization 72° C., 5 min7. termination 4° C.

GSP (SEQ ID NO: 120): TCT CTT TTT CGT GCT GCT CCA GCC GAT (Are 297)PCR program: 10 min. 95° C.

-   -   1 min. 95° C. (40 cycles)    -   1 min. 65° C.    -   2 min. 72° C.    -   10 min. 72° C. interval 4° C.        PCR apparatus: Biometra Trio Thermoblock

First PCR on the RACE library moss with AP1 and GSP, when size correctPCR with nested AP2 and GSP, positives are cloned into pCRII-TOPO-TAcloning vector for sequencing purposes.

Example 14 Expression of Thraustochytrium LPAAT (ThLPAAT) in Yeast

To detect the functionality of ThLPAAT, the coding region of the cDNAwas, in a first approach, cloned into a yeast expression vector andexpressed in S. cerevisiae. The LPAAT produced in the yeast should bedetected added via acyltransferase activity in microsomal fractions.

All solid and liquid media for yeast were prepared by protocols ofAusubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1995).

The ThLPAAT cDNA was excised from the vector pGEM-T by a restrictiondigest with HindIII/BamHI, cloned into the HindIII/BamHI-cut shuttlevector pYES2 (Invitrogen, Carlsbad, USA), and the resulting vectorpYES2-ThLPAAT was transformed into E. coli XL1 Blue. With the aid of theLiAc method, pYES2-ThLPAAT was transformed into S. cerevisiae INCSc1(Invitrogen, Carlsbad, USA), where the expression of the ThLPAAT cDNAwas under the control of the GAL1 promoter.

The expression of ThLPAAT in S. cerevisiae INVSc1 was carried out by amodified method of Avery et al. (Appl. Environ. Microbiol., 62, 1996:3960-3966) and Girke et al. (The Plant Journal, 5, 1998: 39-48). Toprepare a starter culture, 20 ml of SD medium supplemented with glucoseand amino acid solution, but without histidine, were inoculated with anindividual yeast colony and incubated overnight at 30° C. at 140 rpm.The cell culture was washed twice by centrifugation and resuspended inSD medium without supplements and without sugar. The washed cells wereused to inoculate a main culture to an OD₆₀₀ of from 0.1 to 0.3. Themain culture was grown in 25 ml of SD medium supplemented with 2% (w/v)galactose, amino acid solution without histidine, 0.02% linoleic acid(2% strength stock solution in 5% Tergitol NP40), 10% Tergitol NP40 for72 hours at 30° C. The main culture was harvested by centrifugation. Thecell pellet was frozen at −20° C. and then lyophilized for approximately18 hours.

After expression of the construct pYES2-ThLPAAT in yeast, no activeprotein was purified, nor did the subcellular fractions from thedifferent transgenic cells show higher LPAAT activities than thecorresponding control fractions.

To increase the solubility of the expressed protein, a further constructpDest15-GST-ThLPAAT (pDest15 vektor from Invitrogen) was generated viathe Gateway reaction. To this end, the following primers weresynthesized following the manufacturer's instructions:

5′ primer att1ThLPAAT (SEQ ID NO: 121):GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAGCGCGTGGACGAGG GCC 3′primer att2ThLPAAT (SEQ ID NO: 122):GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGTGGTGGTGGTGGTGGTGCAAGAGGTCAGGTCGGACGTAC

These primers were used to carry out the following PCR reaction:

PCR Standard Mix (for Taq Polymerase)

5 μl 10×PCR buffer (100 mM Tri-HCl, pH 8.3; 15 mM MgCl₂, 500 mM KCl)1 μl dNTP mix (10 mM dATP, dGTP, dTTP and dCTP)1 μl primer 1 (30 μM)1 μl primer 2 (30 μM)1 U Taq polymerase50-100 ng pYES2-ThLPAATmake up to 50 μl with distilled waterPCR program: 2 min. 95° C.

-   -   1 min. 95° C. (30 cycles)    -   1 min. 65° C.    -   2 min. 72° C.    -   10 min. 72° C. interval 4° C.

PCR Apparatus: Biometra Trio Thermoblock

The PCR product was transferred into the vector pDONOR221 by Gatewayreaction (BP reaction; Invitrogen) following the manufacturer'sinstructions, and the sequence was verified by sequencing. In a nextstep, the ThLPAAT sequence was then transferred into the vector pDES15by the LR reaction and employed for expression in E. coli BL21 cells.The ThLPAAT sequence was attached to the open reading frame of theglutathione-S transferase (GST) encoded in the plasmid, in accordancewith the manufacturer's instructions. This gave rise to a fusion proteinof GST and ThLPAAT.

Expressed protein was detected after expression under standardconditions in E. coli (FIG. 21A) and purified via a glutathione column.

The purified fusion protein showed LPAAT activity, as shown in FIG. 21B.The highest activity was obtained for DHA-CoA (22:6), which makespossible a utilization of this acyltransferase for the production ofPUFA.

FIG. 21A shows the Western blot analyses of the Thraustochytrium LPAATexpressed in E. coli as fusion protein (LPAAT-FP) with N-terminal GSTtag and C-terminal His tag (lines E: 7 μg soluble protein fraction, lineM: size standard). FIG. 21B shows the acyl-CoA specificity of theThraustochytrium LPAAT, expressed as GST fusion protein, in E. coli. Theenzyme assays were determined using 0.4 μg of soluble protein fractionin the presence of 100 mM Tricine-NaOH (pH 8.2), 30 μM1-oleoyl[U-¹⁴C]glycerol-3-phosphate and increasing concentrations of thethioesters detailed.

Example 15 Expression of Shewanella LPAAT

To clone an LPAAT gene from the prokaryotic organism Shewanella, thegenomic DNA from Shewanella hanedai was isolated, partially digestedwith Sau3a and ligated into the vector pUC18. This genomic library wasscreened for LPAAT genes by a PCR using different primer combinations.This method has made it possible to identify a 1486 bp clone whose openreading frame codes for a 25.2 kDa LPAAT protein. The ShLPAAT sequencewas introduced into the vector pQE70 (Qiagen) in accordance with themanufacturer's instructions. The resulting plasmids pQE70-Sh andpQE70-ShHis and the blank vector pQE70 were transformed into E. coliBL21 cells and expressed at 10° C. (FIG. 22A). Active protein wasobtained at this temperature only (FIG. 22B). The membrane fractionswere used for this purpose in the further experiments. In bothexpression forms, this fraction showed a high level of activity withregard to the incorporation of DHA-CoA (22:6-CoA). The highincorporation rate with regard to PUFA acyl-CoA residues is required forthe use for the production of PUFA.

FIG. 22A: shows the Western blot analysis of the Shewanella LPAATexpressed in E. coli as fusion protein with C-terminal His-tag (line E:7 μg of inclusion body fraction, line F: 7 μg of membrane fraction, lineM: size standard). FIG. 22B: shows the functional expression of theShewanella LPAAT in E. coli enzyme assays. The assays were carried outwith extracts (1 μg) from E. coli comprising the blank vector (pQE70) ora Shewanella construct without (pQE-Sh) or with His-Tag sequence at the3′ end (pQE-ShHis) in the presence of 30 μM1-oleoyl[U-¹⁴C]glycerol-3-phosphate and 30 μM of the detailedthioesters.

Example 16 Expression of Mortierella LPAAT (MaLPAAT, MaB4) in Yeast

The MaLPAAT cDNA was amplified via PCR with the stated primersMaLPAAT1.1, the PCR product was cloned into the vector pENTR-SD-D-TOPO(Invitrogen, Carlsbad, USA) in accordance with the manufacturer'sinstructions and transformed into E. coli XL1 Blue. The MaLPAAT fragmentwas transferred from the resulting vector pENTR-SD-D-MaLPAAT via Gatewayreaction in accordance with the manufacturer's instructions (Invitrogen,Carlsbad, USA) into the vector pYES54Dest, resulting in the vectorpYES52Dest-MaLPAAT. PYES52Dest-MaLPAAT was transformed into S.cerevisiae INCSc1 (Invitrogen, Carlsbad, USA) with the aid of the LiAcmethod.

Yeast cells which had been transformed with the plasmidpYES52Dest-MaLPAAT were analyzed as follows:

Yeast colonies which, after transformation, were capable of growing ondropout uracil minimal medium were again streaked on dropout uracilminimal medium and then grown on liquid minimal medium to an OD600 of0.8. This preculture was then used for inoculating the main culturewhich, besides the minimal medium, additionally comprised 2% (w/v)galactose and 250 μM of the fatty acids. After incubation of the mainculture for 24 hours at 30° C., the cells were harvested bycentrifugation (100×g, 10 min, 20° C.) and washed with 100 mM NaHCO₃, pH8.0, in order to remove residual medium and fatty acids. Fatty acidmethyl esters (FAMEs) were prepared from the yeast cell sediments byacid methanolysis. To this end, the cell sediments were incubated for 1hour at 80° C. with 2 ml of 1N methanolic sulfuric acid and 2% (v/v)dimethoxypropane. The FAMEs were extracted by two extractions withpetroleum ether (PE). To remove nonderivatized fatty acids, the organicphases were washed in each case once with 2 ml of 100 mM NaHCO₃, pH 8.0,and 2 ml of distilled water. Thereafter, the PE phases were dried withNa₂SO₄, evaporated under argon and taken up in 100 μl of PE. The sampleswere separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 μm,Agilent) in a Hewlett Packard 6850 gaschromatograph equipped with flameionization detector. The conditions for the GLC analysis were asfollows: the oven temperature was programmed from 50° C. to 250° C. withan increment of 5° C./min and finally 10 minutes at 250° C. (holding).

The signals were identified by comparing the retention times withcorresponding fatty acid standards (Sigma).

The methodology is described for example in Napier and Michaelson, 2001,Lipids. 36(8):761-766; Sayanova et al., 2001, Journal of ExperimentalBotany. 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem.Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters.439(3):215-218.

FIG. 23 shows the results of the feeding experiments with the yeastcells which comprise plasmid pYES52Dest-MaLPAAT (MaB4_AT). In FIG. 23,NB, the yeast cultures were fed linoleic acid (18:2 Δ9,12). Incomparison with the control culture (FIG. 23, A), the yeast cells withthe MaLPAAT showed a markedly higher conversion (increased 4-fold) of18:2 into γ-linolenic acid (18:3 Δ6,9,12), and a 3.5-fold increase ofthe fatty acid 20:2 Δ11,14 elongated from 18:2. Analogously, whenfeeding linolenic acid (18:3 Δ9,12,15), a markedly higher conversionrate to give stearidonic acid (18:4 Δ6,9,12,15) and isoarachidonic acid(20:4 Δ8,11,14,17) was observed in comparison with the controls (FIG.24).

Besides this activity, an enhanced conversion of 16:1 Δ9 (endogenousfatty acid in yeast) to give cis-vaccenic acid (18:1 Δ11) was observedin both feeding experiments.

FIG. 25 and FIG. 26 show that the observed enhanced conversion rates ofthe substrates by the desaturase and the elongase also leads to anincrease in the polyunsaturated fatty acids in the neutral lipid (oil).After the yeasts had been fed linoleic or linolenic acid, the yeastcells were extracted in chloroform:methanol (2:1) and applied to asilica thin-layer plate (Machery&Nagel, Düren). The thin-layer plate wasincubated for 45 minutes with chloroform-methanol-H2O (65:25:4) in achamber. In doing so, the neutral lipids (triacylglycerides) migratewith the solvent front. After the incubation had ended, the neutrallipids were scraped off from the plate, extracted withchloroform:methanol and analyzed by gas chromatography.

The increase in the conversion rate of PUFAs, which had been observedfor the total extracts, was clearly also monitored in the neutrallipids. As regards the feeding of linoleic acid (FIGS. 25 A and B), a2-fold increase in the conversion of linoleic acid into γ-linolenic acid(18:3 Δ6,9,12) and a 3-fold increase in the 20:2 49,12 content wasobserved. The feeding of linolenic acid (FIGS. 26, C and D) gave similardata (conversion of 18:3 into 18:4 3-fold, of 18:3 into 20:3 3-fold).

Thus, it was demonstrated that the increase in the PUFA content as theresult of MaLPAAT leads to an increase in PUFAs in the oil (neutrallipids) of the yeasts.

Example 16 Plasmids for Plant Transformation

Binary vectors such as pBinAR can be used for transforming plants(Höfgen and Willmitzer (1990) Plant Science 66: 5221-230). The binaryvectors can be constructed by ligating the cDNA in sense or antisenseorientation into T-DNA. 5′ of the cDNA, a plant promoter activates thetranscription of the cDNA. A polyadenylation sequence is located 3′ ofthe cDNA.

Tissue-specific expression can be achieved using a tissue-specificpromoter. For example, seed-specific expression can be achieved bycloning the napin or the LeB4 or USP promoter 5′ of the cDNA. Any otherseed-specific promoter element can also be used. The CaMV-355 promotercan be used for obtaining constitutive expression in all of the plant.The expressed protein can be targeted into a cellular compartment usinga signal peptide, for example for plastids, mitochondria or theendoplasmic reticulum (Kermode (1996) Crit. Rev. Plant Sci. 15:285-423). The signal peptide is cloned 5′ in the reading frame with thecDNA in order to obtain the subcellular localization of the fusionprotein.

Example 17 Transformation of Agrobacterium

The Agrobacterium-mediated transformation of plants can be carried outfor example using the Agrobacterium tumefaciens strain GV3101 (pMP90)(Koncz and Schell (1986) Mol. Gen. Genet. 204: 383-396) or LBA4404(Clontech). The transformation can be carried out by standardtransformation techniques (Deblaere et al. (1984) Nucl. Acids. Res. 13:4777-4788).

Example 18 Plant Transformation and Expression of PUFA-SpecificAcyltransferases in Plants

The expression of LCPUFA-specific acyltransferases in transgenic plantsis advantageous in order to increase the LCPUFA content in these plants.To this end, the acyltransferase cDNAs according to the invention werecloned into binary vectors and transferred into Arabidopsis thaliana,Nicotiana tabacum, Brassica napus and Linum usitatissimum viaAgrobacterium-mediated DNA transfer. Here, the expression of theacyltransferase cDNA was under the control of the constitutive CaMV 35 Spromoter or the seed-specific USP promoter.

Especially preferred in this context are transgenic plants which alreadyexpress the desaturases and elongases required for the synthesis ofLCPUFAs and which produce small amounts of these LCPUFAs.

The expression vectors used were the vector pBinAR (Höfgen andWillmitzer, Plant Science, 66, 1990: 221-230) or the pBinAR derivativepBinAR-USP, in which the CaMV 35 S promoter had been replaced by the V.faba USP promoter. The vectors pGPTV and pGPTV-USP were also used. Tocarry out the recloning step, it was necessary to excise the CalDes cDNAfrom the vector pGEM-T and clone it into pBinAR or pBinAR-USP. A furtherbinary vector which was used was pSUN.

The resulting binary vectors with acyltransferase genes were transformedinto Agrobacterium tumefaciens (Höfgen and Willmitzer, Nucl. Acids Res.,16, 1988: 9877). A. thaliana was transformed by means of floral dip(Clough and Bent, Plant Journal, 16, 1998: 735-743), and N. tabacum viacoculturing tobacco leaf segments with transformed A. tumefaciens cells,and linseed and oilseed rape by coculturing hypocotyl segments withtransformed A. tumefaciens cells.

The expression of the acyltransferase genes in transgenic Arabidopsis,tobacco, oilseed rape and linseed plants was analyzed via Northern blotanalysis. Selected plants were analyzed for their content in punicicacid or other conjugated fatty acids such as CLA in the seed oil.

To obtain seed-specific expression of PuFADX and PuFAD12, it is alsopossible to use the napin promoter analogously to the USP promoter.

The Agrobacterium-mediated transformation of plants can be carried outusing standard transformation and regeneration techniques (Gelvin,Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual,2^(nd) Ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., RingbucZentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R.,Thompson, John E., Methods in Plant Molecular Biology and Biotechnology,B. Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).

For example, oilseed rape can be transformed by cotyledon or hypocotyltransformation (Moloney et al., Plant Cell Report 8 (1989) 238-242; DeBlock et al., Plant Physiol. 91 (1989) 694-701). The use of antibioticsfor the selection of Agrobacteria and plants depends on the binaryvector and the agrobacterial strain used for the transformation. Oilseedrape is usually selected using kanamycin as selectable plant marker. Theagrobacterium-mediated gene transfer into linseed (Linum usitatissimum)can be carried out for example using a technique described by Mlynarovaet al. (1994) Plant Cell Report 13: 282-285.

Soybean can be transformed for example using a technique described inEP-A-0 0424047 (Pioneer Hi-Bred International) or in EP-A-0 0397687,U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770 (University Toledo).The transformation of plants using particle bombardment, polyethyleneglycol-mediated DNA uptake or via the silicon carbonate fiber techniqueis described for example by Freeling and Walbot “The maize handbook”(1993) ISBN 3-540-97826-7, Springer Verlag New York).

Example 19 Analysis of the Expression of a Recombinant Gene Product in aTransformed Organism

The activity of a recombinant gene product in the transformed hostorganism was measured at the transcriptional and/or the translationallevel.

A suitable method for determining the amount of transcription of thegene (an indication of the amount of RNA available for the translationof the gene product) is to carry out a Northern blot as detailedhereinbelow (reference, see Ausubel et al. (1988)

Current Protocols in Molecular Biology, Wiley: New York, or theabove-mentioned examples section), where a primer which is such that itbinds to the gene of interest is labeled with a detectable label(usually a radioactive or chemiluminescent label) so that, when thetotal RNA of a culture of the organism is extracted, separated on a gel,transferred to a stable matrix and incubated with this probe, thebinding, and the degree of the binding, of the probe indicates thepresence and also the amount of the mRNA for this gene. This informationindicates the degree of the transcription of the transformed gene.Cellular total RNA can be prepared from cells, tissues or organs using aplurality of methods, all of which are known in the art, such as, forexample, the method described by Bormann, E. R., et al. (1992) Mol.Microbiol. 6:317-326.

Northern Hybridization:

To carry out the RNA hybridization, 20 μg of total RNA or 1 μg ofpoly(A)⁺ RNA were separated as described in Arnasino (1986, Anal.Biochem. 152, 304) by means of gel electrophoresis in agarose gels witha strength of 1.25% using formaldehyde, transferred by capillaryattraction using 10×SSC to positively charged nylon membranes (HybondN⁺, Amersham, Brunswick), immobilized by means of UV light andprehybridized for 3 hours at 68° C. using hybridization buffer (10%dextran sulfate weight/vol., 1 M NaCl, 1% SDS, 100 mg herring spermDNA). The DNA probe was labeled with the Highprime DNA labeling kit(Roche, Mannheim, Germany) during the prehybridization step, usingalpha-32P-dCTP (Amersham, Brunswick, Germany). The hybridization wascarried out at 68° C. overnight in the same buffer after addition of thelabeled DNA probe. The wash steps were carried out twice for 15 minutesusing 2×SSC and twice for 30 minutes using 1×SSC, 1% SDS, at 68° C. Thesealed filters were exposed at −70° C. for a period of from 4 hours to 3days.

To analyze the presence or the relative amount of protein translated bythis mRNA, it is possible to employ standard techniques such as aWestern blot (see, for example, Ausubel et al. (1988) Current Protocolsin Molecular Biology, Wiley: New York). In this method, the cellulartotal proteins are extracted, separated by means of gel electrophoresis,transferred to a matrix such as nitrocellulose, and incubated with aprobe, such as an antibody, which binds specifically to the desiredprotein. This probe is usually provided with a chemiluminescent orcolorimetric label which is easy to detect. The presence and the amountof the observed labeling indicates the presence and the amount of thedesired mutated protein which is present in the cell.

Example 20 Analysis of the Effect of the Recombinant Proteins on theProduction of the Desired Product

The effect of the genetic modification in plants, fungi, algae,ciliates, or on the production of a desired compound (such as a fattyacid) can be determined by growing the modified microorganisms or themodified plant under suitable conditions (like those described above)and analyzing the medium and/or the cellular components for theincreased production of the desired product (i.e. of lipids or a fattyacid). These analytical techniques are known to the skilled worker andcomprise spectroscopy,

thin-layer chromatography, various types of staining methods, enzymaticprocesses, microbiological processes and analytical chromatography suchas high-performance liquid chromatography (see, for example, Ullmann,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III:

“Product recovery and purification”, pp. 469-714, VCH: Weinheim; Belter,P. A., et al. (1988) Bioseparations: downstream processing forBiotechnology, John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S.(1992) Recovery processes for biological Materials, John Wiley and Sons;Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations, in:Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp.1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

In addition to the abovementioned processes, plant lipids are extractedfrom plant material as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986) AnalyticBiochemistry 152:141-145. Qualitative and quantitative lipid or fattyacid analysis is described by Christie, William W., Advances in LipidMethodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2);Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (OilyPress Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

Besides measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for the production of the desired compound, such as intermediateand secondary products, in order to determine the overall efficiency ofthe production of the compound. The analytical methods comprisemeasuring the amounts of nutrient in the medium (for example sugars,hydrocarbons, nitrogen sources, phosphate and other ions), measuring thebiomass composition and the growth, analysis of the production of commonmetabolites of biosynthetic pathways and measuring gases which aregenerated during the fermentation. Standard methods for thesemeasurements are described in Applied Microbial Physiology; A PracticalApproach, P. M. Rhodes and P. F. Stanbury, Ed., IRL Press, 10 pp.131-163 and 165-192 (ISBN: 0199635773) and references cited therein.

One example is the analysis of fatty acids (abbreviations: FAMEs, fattyacid methyl esters; GC-MS, gas-liquid chromatography-mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

The unambiguous detection for the presence of fatty acid products can beobtained by means of analyzing recombinant organisms by analyticalstandard methods: GC, GC-MS or TLC, as described repeatedly by Christieand the references cited therein (1997, in: Advances on LipidMethodology, Fourth Ed.: Christie, Oily Press, Dundee, 119-169; 1998,Gas-chromatography/mass spectrometry methods, Lipids 33:343-353).

The material to be analyzed can be disrupted by sonication, grinding ina glass mill, liquid nitrogen and grinding, or via other suitablemethods. After disruption, the material must be centrifuged. Thesediment is resuspended in distilled water, heated for 10 minutes at100° C., cooled on ice and recentrifuged, followed by extraction in 0.5M sulfuric acid in methanol supplemented with 2% dimethoxypropane for 1hour at 90° C., which leads to hydrolyzed oil and lipid compounds, whichgive transmethylated lipids. These fatty acid methyl esters areextracted in petroleum ether and finally subjected to GC analysis usinga capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25micrometers, 0.32 mm) at a temperature gradient between 170° C. and 240°C. for 20 minutes and 5 minutes at 240° C. The identity of the resultingfatty acid methyl esters must be defined using standards which areavailable from commercial sources (i.e. Sigma).

In the case of fatty acids for which no standards are available, theidentity must be shown via derivatization and subsequent GC-MS analysis.For example, the localization of fatty acids with triple bond must beshown via GC-MS after derivatization with 4,4-dimethoxyoxazolinederivatives (Christie, 1998, see above).

EQUIVALENTS

The skilled worker recognizes, or will find, a multiplicity ofequivalents of the specific embodiments according to the inventiondescribed herein by simply using routine experiments. The patent claimsare intended to encompass these equivalents.

We claim:
 1. A process for the production of polyunsaturated fatty acidsin an organism, which comprises: a) introducing, into an organism, atleast one nucleic acid sequence comprising: i) the sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO:36; ii) a sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37; or iii) a sequenceencoding a polypeptide having at least 40% sequence identity to theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37 and havingan equivalent enzymatic activity as the polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37; and b)culturing and harvesting said organism.